Genetically stable recombinant modified vaccinia ankara (rMVA) vaccines and methods of preparation thereof

ABSTRACT

A vaccine comprising an immunologically effective amount of recombinant modified vaccinia Ankara (rMVA) virus which is genetically stable after serial passage and produced by a) constructing a transfer plasmid vector comprising a modified H5 (mH5) promoter operably linked to a DNA sequence encoding a heterologous foreign protein antigen, wherein the expression of said DNA sequence is under the control of the mH5 promoter; b) generating rMVA virus by transfecting one or more plasmid vectors obtained from step a) into wild type MVA virus; c) identifying rMVA virus expressing one or more heterologous foreign protein antigens using one or more selection methods for serial passage; d) conducting serial passage; e) expanding an rMVA virus strain identified by step d); and f) purifying the rMVA viruses from step e) to form the vaccine. One embodiment is directed to a fusion cytomegalovirus (CMV) protein antigen comprising a nucleotide sequence encoding two or more antigenic portions of Immediate-Early Gene-1 or Immediate-Early Gene-2 (IEfusion), wherein the antigenic portions elicit an immune response when expressed by a vaccine.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.12/795,621, filed Jun. 7, 2010, which claims priority to U.S.Provisional Application No. 61/184,767, filed Jun. 5, 2009, both ofwhich are incorporated herein by reference in their entireties,including drawings.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The invention was made with Government support under Grant No. CA030206awarded by the Public Health Service, Grant Nos. CA077544 and CA114889awarded by the National Cancer Institute and Grant No. AI062496 awardedby the National Institute of Allergy and Infectious Disease. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The invention relates to genetically engineered modified vaccinia Ankara(MVA) or recombinant MVA (rMVA) vaccines with improved stability duringextended passage. Specifically, the invention relates to geneticallystable rMVA vaccines expressing cytomegalovirus (CMV) antigens such asan IEfusion protein. The invention also relates to methods for improvinggenetic stability and maintaining immunogenicity of rMVA vaccines afterserial passage. The invention further relates to methods for thepreparation of the rMVA vaccines.

(2) Description of the Related Art

Modified vaccinia Ankara (MVA) is a genetically engineered, highlyattenuated strain of vaccinia virus that does not propagate in mostmammalian cells (Daftarian et al. 2005)). This property minimallyimpacts viral or foreign gene expression because the ability of MVA toreplicate in mammalian cells is blocked at late stage viral assembly.MVA also has a large foreign gene capacity and multiple integrationsites, two features that make it a desirable vector for expressingvaccine antigens. MVA has a well-established safety record andversatility for the production of heterologous proteins (Drexler et al.2004; Ramirez et al. 2000; Stickl et al. 1974; Stittelaar et al. 2001;Werner et al. 1980). In fact, MVA-based vaccines for treatment ofinfectious disease and cancer have been developed and reached Phase I/IIclinical trials (Acres 2007; Cosma et al. 2003; Gilbert et al. 2006;Peters et al. 2007; Rochlitz et al. 2003).

MVA has an extensive history of successful delivery into rodents, Rhesusmacaques, and other non-human primates, and more recently as a clinicalvaccine in cancer patients (Gilbert et al. 2006; Peters et al. 2007;Rochlitz et al. 2003). MVA is avirulent because of the loss of twoimportant host-range genes among 25 mutations and deletions thatoccurred during its repeated serial passage in chicken cells (Antoine etal. 1998; Meyer et al. 1991). In contrast to NYVAC (attenuatedCopenhagen strain) and ALVAC (host-range restricted Avipox), both earlyand late transcription are unimpaired making MVA a stronger vaccinecandidate (Blanchard et al. 1998; Carroll et al. 1997a; Carroll et al.1997b; Zhang et al. 2007). Studies in rodents and macaques affirm thesafety of MVA, including protection against more virulent forms of poxviruses in challenge models and lack of persistence three months beyondinitial dosing administration (deWaal et al. 2004; Earl et al. 2007;Hanke et al. 2005). Similarly, a therapeutic vaccination with MVAexpressing HIV-nef demonstrated its safety in HIV-infected individuals(Cosma et al. 2003). Finally, MVA immunizations of malaria patientscoinfected with HIV and/or TB confirm the safety of the vector and itsability to partially protect against a heterologous malaria strain(Gilbert et al. 2006; Moorthy et al. 2003).

These properties make MVA appealing as a vaccine vector for CMV antigensin individuals who are both severely immunosuppressed and experiencingadditional complications such as malignancy or organ failure, therebyrequiring a transplant. CMV infection is an important complication oftransplantation procedures and affects a wide variety of individualsincluding newborns and HIV patients with advanced disease (Pass et al.2006; Sinclair et al. 2006; Zaia 2002). Individuals who are previouslyCMV-infected or receiving a CMV-infected solid organ or stem cellallograft are candidates for a vaccine strategy that targets thecellular reservoir of the virus (Zaia et al. 2001).

Several antigens have been identified as being associated withprotective immunity against CMV in transplant recipients. These includethe tegument protein pp65 (UL83) and the immediate-early 1 (IE1 orUL123) global gene expression regulator (Boeckh et al. 2006; Cobbold etal. 2005; Cwynarski et al. 2001; Einsele et al. 2002; Gratama et al.2001). In addition, a recent proteomic study of the whole CMV genomedivided into overlapping peptides showed that pp65 stimulatessubstantial levels of both CD8+ and CD4+ T cells, while IE1 mainlystimulates CD8+ T cells, and the related IE regulator referred to as IE2(UL122) stimulates a vigorous CD8+ and a smaller CD4+ T cell memoryresponse by a large percentage of asymptomatic CMV-positive adults(Sylwester et al. 2005). Other antigens are also recognized with robustcellular immune responses, but the evidence for these three antigens tobe highly recognized in a majority of CMV-infected healthy subjects andtransplant patients (Gallez-Hawkins et al. 2005) is compelling andjustifies their inclusion into a vaccine to prevent viremia and controlinfection.

Because MVA only replicates in the cytoplasm of cells with its ownvaccinia transcriptional system (which does not recognize other viraland cellular promoters), vaccinia viral promoters are used to directforeign antigen gene expression efficiently in recombinant MVA (rMVA)systems. There are two types of vaccinia promoters widely used to directforeign gene expression in recombinant MVA. One is pSyn, which containsboth vaccinia early and late promoter sequences optimized for high levelprotein expression (Chakrabarti et al. 1997). The other is modified H5promoter (mH5), which contains both native early and late vacciniapromoter regions. pSyn has stronger overall promoter activity than mH5,but the early activity of the mH5 promoter is three- to five-foldstronger than the pSyn series.

It has been reported that in vitro expression levels of foreign antigensby an rMVA vaccine are correlated with the rMVA vaccine's immunogenicity(Wyatt et al. 2008b). For example, mice immunized with the rMVAsexpressing high level of human immunodeficiency virus (HIV) Env proteinhad about 15-fold higher titers of Env antibodies and several foldhigher frequencies of Env-specific CD8+ and CD4+ T cells than miceimmunized with rMVAs expressing low level of Env (84). However, afterserial passage, the foreign antigen expression may be reduced andrendered unstable, which can result in diminished immunogenicity.

Thus, while MVA is an attractive viral vector for recombinant vaccinedevelopment, genetic instability and diminished immunogenicity aresignificant concerns after serial passage. The beneficial effect of highantigen expression levels and improved immunogenicity can be limited bythe tendency of rMVA to delete genes unnecessary for its lifecycle.Previous reports suggest that instability of rMVA vaccines may berelated to toxicity of foreign protein in the gene region in which it isinserted or the promoter that controls foreign protein expression (Timmet al. 2006; Wyatt et al. 2008a). For example, rMVA viruses expressingHIV Env protein and other rMVAs were found to have non-expressing mutantvirus accumulation after serial passage (Wyatt et al. 2008a). rMVAexpressing hemagglutinin-neuraminidase (HN) glycoproteins under controlof pSyn was previously reported to replicate poorly (Wyatt et al. 1996).The non-expressing mutants and poor replications of rMVAs were reportedto be likely due to toxicity of the expression of foreign proteins(Wyatt et al. 2008a; Wyatt et al. 1996). However, an rMVA expressing amutated truncation of Env is found to have enhanced genetic stabilityand immunogenicity relative to rMVAs expressing a full-length Env (Wyattet al. 2008a). Thus, a higher expression level of foreign antigensdriven by a strong promoter in rMVA vaccines does not always result inhigher immunogenicity after serial passage. Genetic instability anddiminished immunogenicity after serial passage have not been fullyunderstood.

It will be advantageous to develop an rMVA vaccine with stableexpression of foreign protein antigens and immunogenicity after serialpassage, which will enable the use of MVA as a clinical vector for abroader portfolio of infectious pathogens and cancer antigens.

SUMMARY

One embodiment is directed to a fusion cytomegalovirus (CMV) proteinantigen comprising a nucleotide sequence encoding two or more antigenicportions of Immediate-Early Gene-1 or Immediate-Early Gene-2 (IEfusion),wherein the antigenic portions elicit an immune response when expressedby a vaccine. In one aspect, the IEfusion nucleotide sequence is SEQ IDNO:11.

One embodiment is directed to a vaccine comprising an immunologicallyeffective amount of recombinant modified vaccinia Ankara (rMVA) viruswhich is genetically stable after serial passage and produced by a)constructing a transfer plasmid vector comprising a modified H5 (mH5)promoter operably linked to a DNA sequence encoding a heterologousforeign protein antigen, wherein the expression of said DNA sequence isunder the control of the mH5 promoter; b) generating rMVA virus bytransfecting one or more plasmid vectors obtained from step a) into wildtype MVA virus; c) identifying rMVA virus expressing one or moreheterologous foreign protein antigens using one or more selectionmethods for serial passage; d) conducting serial passage; e) expandingan rMVA virus strain identified by step d); and f) purifying the rMVAviruses from step e) to form the vaccine.

Another embodiment is directed to a method of modifying an immuneresponse in a subject by administering a vaccine composition asdescribed above to the subject. In one aspect, the subject is a human.

Yet, another embodiment is directed to a method for producing agenetically stable rMVA vaccine, comprising a) constructing a transferplasmid vector comprising a modified H5 (mH5) promoter operably linkedto a DNA sequence encoding a heterologous foreign protein antigen,wherein the expression of said DNA sequence is under the control of themH5 promoter; b) generating rMVA virus by transfecting one or moreplasmid vectors obtained from step a) into wild type MVA virus; c)identifying rMVA virus expressing one or more heterologous foreignprotein antigens using one or more selection methods for serial passage;d) conducting serial passage; e) expanding an rMVA virus strainidentified by step d); and f) purifying the rMVA viruses from step e) toform the vaccine; wherein the expression and immunogenicity of saidforeign protein antigens are stable after serial passage in the rMVAvaccine obtained from step e).

In some aspects of some embodiments, at least one of the foreign proteinantigens is a cytomegalovirus (CMV) antigen. In further aspects, the CMVantigen is selected from the group consisting of pp65, CMV pp150, IE1,IE1 exon 4 (IE1/e4), IEfusion, glycoprotein B (gB) and antigenicfragments thereof.

In other aspects of some embodiments, the identification of rMVA viruscarrying the MVA virus vector is accomplished by one or more gene-inselection methods, one or more gene-out selection methods, or acombination of gene-in and gene-out selection methods.

In other aspects of some embodiments, serial passage is at least 10passages.

Another embodiment is directed to an rMVA virus strain comprising anucleotide sequence encoding a modified H5 (mH5) promoter operablylinked to one or more heterologous foreign protein antigens, wherein atleast one of the foreign protein antigens is an IEfusion, said IEfusioncomprising a nucleotide sequence encoding two or more antigenic portionsof Immediate-Early Gene-1 or Immediate-Early Gene-2, wherein theantigenic portions elicit an immune response when expressed by avaccine. In one aspect, the nucleotide sequence of IEfusion is SEQ IDNO:11.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic map of the pp65 and IE1/e4 gene expressioncassette of pSyn-pp65-IE1/e4-MVA generated by homologous recombination.

FIG. 1B illustrates Western blot (WB) detection of pp65 and IE1 exon4expression levels of pSyn-pp65-IE1/e4-MVA after serial passages 1-10.The top panel shows a membrane blotted with mAb28-103 specific for pp65;the middle panel shows a membrane blotted with p63-27 specific forIE1/e4, and the bottom panel shows a membrane blotted with mAB 19C2 thatdetects VV-BR5.

FIG. 1C illustrates Western blot (WB) detection of pp65 expression of 18pSyn-pp65-IE1/e4-MVA individual isolates. Each lane represents a singleindividual isolate from passage 10. Samples #4, #6, #7 and #13 markedwith a star were selected for viral genomic DNA extraction and Southernblot analysis as described below.

FIG. 2A is a series of Western blots detecting pp65 and IE1 exon4protein expression of selected individual isolates of pSyn-pp65-IE1exon4-MVA. FIG. 2A, panel (i) was blotted with mAb 28-103 specific forpp65; FIG. 2A, panel (ii) was blotted with p63-27 specific for IE1 exon4and FIG. 2A, panel (iii) was blotted with mAb specific for vacciniaviral protein. FIG. 2B is a Southern blot detecting pp65 and IE1 exon4gene insertion of selected individual isolates of pSyn-pp65-IE1/e4-MVA.MVA viral genomic DNA was digested with restriction enzymes to excise3.9 Kb fragments of pp65-IE1 gene expression cassettes, separated by 1%agarose gel and transferred to nylon membrane filter. This filter washybridized with the ³²P-radiolabeled DNA probe specific for both pp65and IE1 exon4 gene and exposed to x-ray film. Lanes 1 and 2 in FIGS. 2Aand 2B are two individual isolates selected randomly from passage 1 ofpSyn-pp65-IE1/e4-MVA. Lanes 3 and 4 of FIGS. 2A and 2B are the twoindividually isolates of #4 and #6 marked with * from FIG. 1C with noexpression of pp65 and IE1 exon4. Lanes 5 and 6 of FIGS. 2A and 2B arethe two individual isolates #7 and #13 marked with * in FIG. 1C withpp65 and IE1 exon4 protein expression levels.

FIG. 3 is a bar graph showing the immunogenicity of pSyn-pp65-IE1/e4-MVApassage 1 and passage 10 immunized HHD II mice (HLA A2.1). Averagelevels of IFN-γ producing specific for the CMV pp65- or IE1-A2 epitope(x axis) for all immunized mice is shown in Y-axis. IFN-γ producing CD8⁺T-cells to mock during the ICS procedure were subtracted. Error barsrepresent the SEM for all immunized mice.

FIG. 4A is a bar graph showing data related to the genetic stability ofpSyn-pp65-IE1/e4-MVA at serial passages P0-P10 as determined by qPCR.pSyn-pp65-IE1/e4-MVA genomic DNA was extracted as described in Example8. pSC11 plasmid containing CMV genes (pp65, IE1/e4 and IE2/e5) was usedto prepare absolute standards. The qPCRs were performed using primersspecific for pp65, IE1 exon4 and TK gene. The copy numbers for pp65gene, IE1 gene and MVA backbone copies were calculated using ABIsoftware (SDS3.2) and the genetic stability of the mH5-pp65-IEfusion-MVAwas determined by computing the ratio of the pp65 gene insert and theMVA backbone or the ratio of the IE1 exon4 gene insert and the MVAbackbone as indicated in Y-axis. The ratio at passage 1 was normalizedto 1 and each consecutive passage was normalized based on passage 1. TheqPCR for each DNA sample were performed three times independently induplicate. The average ratio and error bar shown in the figure representthree independent determinants.

FIG. 4B is a bar graph showing data related to the genetic stability ofpSyn-pp65-IEfusion-MVA at serial passages P0-P5 as determined by qPCR.The copy numbers for pp65 gene, IEfusion gene and MVA backbone wereanalyzed using ABI software (SDS3.2) and the genetic stability of themH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65gene insert and the MVA backbone or the ratio of the IEfusion geneinsert and the MVA backbone. The ratios at passage 1 for pp65 and IE1exon4 gene were normalized to 1. The qPCR for each DNA sample wereperformed for three times independently in duplicates and average ratioand error bar shown in FIG. 4B represent three independent determinants.

FIG. 5A is a schematic representation of the insertion sites for thetransfer or shuttle plasmids to generate mH5-pp65-IEfusion-MVA.

FIG. 5B is a bar graph showing quantitative PCR results relating to thegenetic stability of 10 serial passages of mH5-pp65-MVA. Recombinant MVAwas generated using shuttle plasmids that had the mH5 promoter directingthe transcription of pp65. mH5-pp65-MVA viral genomic DNA was extractedand qPCR was performed using pp65, and TK specific primers as describedabove. The copy numbers for pp65 gene and MVA backbone were analyzedusing ABI software (SDS3.2) and the genetic stability of themH5-pp65-IEfusion-MVA was determined by computing the ratio of the pp65gene insert and the MVA backbone. The ratios at passage 1 werenormalized to 1. The qPCR for each DNA sample were performed three timesindependently in duplicate. The average ratio and error bars representthree independent determinants. No significant changes were seen in theratio of CMV gene:MVA backbone genomic copy number during serialpassage. The results of immunogenicity measurements in the HHD II (HLAA2.1) mouse were superior to that observed with similar virusesemploying the pSyn promoter.

FIG. 5C is a bar graph showing quantitative PCR results relating to thegenetic stability of 10 serial passages of mH5-pp65-IEfusion-MVA.mH5-pp65-IEfusion-MVA genomic DNA was extracted and qPCR was performedusing pp65, IEfusion and TK specific primers as described in theExamples below. The copy numbers for pp65 gene, IEfusion gene and MVAbackbone were analyzed using ABI software (SDS3.2) and the geneticstability of the mH5-pp65-IEfusion-MVA was determined by computing theratio of the pp65 gene insert and the MVA backbone or the ratio of theIEfusion gene insert and the MVA backbone. The ratios at passage 1 forpp65 and IE1/e4 gene were normalized to 1.

FIG. 5D is a bar graph, similar to FIG. 5C, except the 10 serialpassages were conducted on CEF and results shown are computed using pp65and TK-specific primers. The qPCR for each DNA sample were performed forthree times independently in duplicates and the ratios and error barsshown in the figure represent an average of three independentdeterminants.

FIG. 6A is a bar graph showing the immunogenicity ofmH5-pp65-IEfusion-MVA of passage 1 and 7 using human peripheral bloodmononuclear cells (PBMC). PBMCs from healthy donors who were ex vivopositive responders to CMV antigens (Wang et al. 2008) were incubatedwith antigen presenting cells infected with either passage 1 or passage7 of mH5-pp65-IEfusion-MVA for 7 days followed by overnight incubationwith diluent (mock), pp65, IE1 or IE2 peptide libraries in the presenceof brefeldin A. Cells were then harvested and stained with anti-humanCD8 or CD4, permeabilized and stained with anti-human IFN-γ antibodiesand evaluated by flow cytometry. Average percentages of IFN-γ producingCD8 or CD4 T cells are shown (N=4). Error bars represent standarddeviation.

FIG. 6B is a bar graph showing the immunogenicity ofmH5-pp65-IEfusion-MVA of passage 1 and 7 in HHD II mice (HLA A2.1)Splenocytes from HHD II mice immunized with pSyn-pp65-IE1 exon4-MVA frompassage 1 (P1) or passage 7 (p7) were subjected to in vitro stimulation(IVS) separately with either pp65 A2 or IE1A2 peptides or IE2 peptidelibrary-loaded HLA-A*0201 EBV-lymphoblastoid cells (LCL) derived from ahealthy CMV positive volunteer (La Rosa et al. 2001) for 8 days. AfterIVS, the splenocytes were incubated with mock A2, pp65A2, IEA2 peptidesor IE2 peptide library overnight and harvested for ICC as described inthe examples below. Average levels of CD8+ T-cell IFN-γ productionspecific for the CMV pp65A2, IE1A2 epitopes or IE2 peptide library shown(x-axis) for all immunized mice. IFN-γ production to mock stimulatedcells during the ICS procedure was subtracted. Error bars represent theSEM for all immunized mice.

FIG. 7A is a plasmid map of mH5-pp65-pLW51(GUS) plasmid (SEQ ID NO:9).

FIG. 7B is a plasmid map of mH5-IEfusion-pZWIIA (GUS) plasmid (SEQ IDNO:10).

FIG. 8A is the mH5-IEfusion-pZWIIA (GUS) plasmid DNA sequence (SEQ IDNO:9).

FIG. 8B is the mH5-pp65-GUS-pLW51(GUS) plasmid DNA sequence (SEQ IDNO:10).

FIG. 9A illustrates the genomic structure of the regulatoryimmediate-early genes IE1 and IE2 of HCMV. IE1 is composed of 4 exons(exon1, 2, 3 and 4) indicated by solid dark lines and three introns asindicated by intervening thin lines; IE2 is also composed of 4 exons(exon1, 2, 3 and 5) as indicated by solid dark lines and three intronsas indicated by intervening thin lines.

FIG. 9B illustrates construction of the IEfusion gene. Primers a, b, c,d, e are described in Example 1. IE1/e4 was amplified from the IE1 geneusing primers a and b, and was further extended using primer a and c tointroduce an internal Apa I site, and external Pme I and Asc I sites.IE2/e5 was amplified from the IE2 gene using primers d and e. It wasdigested at the newly created Apa I and synthetic Asc I site. IE1/e4 andIE2/e5 were joined together by ligation preserving the reading frame(shown as SEQ ID NO: 18).

FIG. 9C is a schematic map of IEfusion-pZWIIA and pp65-IEfusion-pZWIIAMVA transfer plasmids. pZWIIA, an ampicillin resistant plasmid (ampshown in light grey) inserts DNA sequence within the boundaries of MVAdeletion II via flanking regions 1 and 2 (FL1, FL2). pZWIIA has twovaccinia synthetic E/L promoters of slightly different sequence,arranged head to head to each drive expression of separate genes.IEfusion gene is driven by pSyn I promoter (Chakrabarti et al. 1997) andpp65 gene is driven by pSyn II promoter (Wyatt et al. 2004) The gusbacterial marker gene, used for identifying recombinant MVA, is flankedby two direct repeat (DR) sequences to facilitate gus gene removal byintragenomic recombination from IEfusion-MVA or pp65-IEfusion-MVA. pp65was not fused to the IEfusion gene in either transfer plasmid.

FIG. 9D illustrates the generation of IEfusion-MVA andpp65-IEfusion-MVA. IEfusion-pZWIIA or pp65-IEfusion-pZWIIA wastransfected into wtMVA infected CEF cells to generate IEfusion-MVA orpp65-IEfusion-MVA via homologous recombination at deletion II whoseflanking region is contained in the plasmid that is homologous to wtMVA.

FIG. 10A is a Western blot (WB) detection of the pp65 protein antigen.Lane 1: CEF cell lysate infected with pp65-rMVA as (+) control; Lanes 2and 3: cell lysate from wtMVA-infected and uninfected CEF as (−)controls; Lane 4: cell lysate of pp65-IEfusion-MVA-infected CEF cells.The WB in Panel A was incubated with mAb 28-103 against pp65.

FIG. 10B is a Western blot (WB) detection of IEfusion protein antigens.Lane 5: cell lysate of CEF infected with rMVA expressing IE1/e4 as (+)control; Lanes 6 and 7: cell lysate from wtMVA-infected and uninfectedCEF as (−) controls; Lane 8: cell lysate of pp65-IEfusion-MVA-infectedCEF cells and Lane 9: cell lysate of IEfusion-MVA-infected CEF cells.The WB incubated with mAb p63-27 against IE1.

FIG. 11 is a bar graph showing the percentage of interferon-gamma(IFN-γ) producing splenocytes specific for pp65, IE1 and IE2 (x axis) inthree HHDII mice immunized with 50 million pfu of pp65-IEfusion-MVA.Grey bars represent pp65-, IE1- and IE2-specific IFN-γ production byCD8+ T cells using either peptide epitopes or libraries (identifiedbelow the x axis) during IVS and ICC stimulations. Unfilled barsrepresent simultaneous pp65-, IE1- and IE2-specific IFN-γ production byCD4+ T cells, following IVS and ICC stimulation with the correspondingCMV libraries indicated below each set of bars. IVS and ICC stimulationconditions are described in Example 1. In all graphs, error barsrepresent standard error of the mean among the immunized mice (N=3). Inall experiments, IFN-γ production to mock stimulated cells wassubtracted. P values indicate statistically significant differencesmeasured by T-test.

FIG. 12 is a bar graph showing the percentage of IFN-γ producingsplenocytes assessed by flow cytometry specific for pp65 (CTL epitope orlibrary), IE1 and IE2 peptide libraries (x axis) in three B7 miceimmunized with 50 million pfu of pp65-IEfusion-MVA, using methods asdescribed in the legend to FIG. 11. In all graphs, error bars representstandard error of the mean among the immunized mice (N=3). In allexperiments, IFN-γ production to mock stimulated cells was subtracted. Pvalues indicate statistically significant differences measured byT-test.

FIG. 13A is a pair of bar graphs showing ex vivo response to pp65, IE1,and IE2 peptide libraries in healthy volunteers. PBMC were obtained fromN=22 healthy volunteers for which we had complete HLA typing. Fivemillion PBMC were divided into four aliquots and were individuallyco-incubated with peptide libraries at 1 μg/ml/peptide in single usealiquots as described in Example 1. PBMC from each individual weretreated in separate cultures with each peptide library at the same time,but not all individuals were evaluated on the same day. Standard gatingprocedures were employed for each individual flow acquisition, such thatconditions were standardized for all evaluations. Separate aliquots fromthe ICC assay were incubated with CD4+, CD8+ or isotype controlantibodies as described in Example 1. The plots show the percentage of TCells that produce IFN-γ for each antigen-specific peptide library.Error bars represent the standard error of the mean calculated usingMicrosoft Excel statistical package.

FIG. 13B is a set of bar graphs showing ex vivo response of PBMC fromHCT recipients. Three examples from each of three separate riskcategories of HCT shown in 3 separate plots (L-R; D+/R+, D−/R+, D−/R+)based on CMV status were evaluated for response against peptidelibraries using the same technical approach as described in A). Datafrom all 3 individuals was averaged in each category, and the error barsrepresent the standard error of the mean.

FIG. 14A is a pair of bar graphs showing that rMVA stimulatesCMV-specific T cells in human PBMC. Using the IEfusion-MVA, as describedin Example 1, APC were infected for 5-6 hours, irradiated, and thencoincubated with unmanipulated PBMC from the autologous individual. Thetime course and conditions of the IVS are described in Example 1. Fourseparate evaluations were conducted with each IVS culture as shown inPanel A. After treatment with the peptide library and ICC was performed,aliquots of PBMC were either stained with CD4 or CD8 antibodies asdescribed in FIG. 13A. Results shown are averages of measurements fromthree CMV-positive individuals selected randomly from a group of blooddonors. Not shown is a comparison with a CMV-negative donor who showedno specific recognition of any of the three peptide libraries after IVSwith IEfusion- and pp65-IEfusion-MVA.

FIG. 14B is a pair of bar graphs showing results of the same protocol asin FIG. 14A, but using the pp65-IEfusion-MVA. PBMC from 8 healthy CMVpositive blood donors were evaluated both ex vivo without manipulationand post-IVS following infection with rMVA as described in FIG. 14A.Statistical differences between ex vivo levels of CMV-specific T Cellsversus post-IVS were calculated as described in Example 1. When a Pvalue is ≦0.05, it is shown above the error bar for each evaluation ofindividual peptide libraries. All methods for IVS, ICC, and flowcytometry are described in Example 1.

FIG. 15 is a set of bar graphs showing that rMVA stimulates CMV-specificT cells in PBMC from HCT recipients. Six examples of patients that wereevaluated for response to peptide libraries shown in FIG. 13B were alsoevaluated after IVS with pp65-IEfusion-MVA. Methods including conditionsfor IVS, post-IVS analysis of cell population, ICC, and flow cytometryare identical as described in FIG. 14. A comparison was made between theex vivo level versus post-IVS for each stimulation, and each category ofdonor and recipient serostatus is shown in 3 separate plots as discussedin the legend to FIG. 13B.

FIG. 16 is a gel illustrating metabolic radio-labeling of CMV-pp65detected by immunoprecipitation after viral infection of CEF cells.mH5-pp65-MVA (lanes 2-5) and pSyn-pp65-MVA (lanes 6-9) viruses were usedto infect primary CEF plated on 60 mm TC dishes at an MOI of 10 for 1hour, followed by depletion of intracellular stores of Met+Cys for 1 h,and labeled with 35S [Met+Cys] for an additional 30 minutes. Excessunlabeled Met+Cys was diluted into fresh medium, and further incubationtimes are indicated in hours (O, 1, 4 and 10) above the gel profile. Atthe conclusion of the “chase” period, cell lysates were made andimmunoprecipitation was conducted as described in the Examples below.The CMV-pp65 antigen detected by the mAb 28-103 is indicated by an arrowto the right and adjacent to the gel profile. The 1st lane at the farleft (Con) represents a control CEF culture that was radio-labeled afterinfection with a gus-MVA virus which expresses the α-glucoronidasebacterial marker without CMV-pp65 (Wang et al. 2007).

FIG. 17 is a scheme of the MVA genome showing deletions and intergenicregions (IGRs). The MVA deletion II insertion site (del II) for themH5-IEfurion-pZWIIA(GUS) shuttle plasmid is shown by the arrow. Theshuttle plasmid has insertion sites for foreign genes (IEFusion)controlled by an mH5 promoter, a marker gene (gus), homologous flankingsequences for recombination (FL1, FL2), ampicillin resistance (amp) anddirect repeats for marker gene removal (DR).

FIG. 18 is a schematic representation of the Deletion II (del II)insertion site on the rMVA Viral DNA for mH5-IEfusion-pZWIIA(GUS).

FIG. 19 is a Western blot verifying IEfusion incorporation byrestriction enzyme digestion (Asc I and Pme I) followed by DNA sequenceanalysis.

FIG. 20 is a qPCR amplification plot for IEfusion. The standard curvewas established with 10²-10⁷ copies of IEfusion. The cycle number isplotted versus the Delta Rn (normalized reporter signal). Delta Rnrepresents the Rn munus the baseline signal established in early PCRcycles. Copy number of R10 isolate sample No. 8B1A1A1A was determined tobe 9×10⁷, which was comparable to the positive control.

FIG. 21 is a Western blot detecting IEfusion antigen in four wt-free MVAisolates (8B1A1A1A (lane 1), 8B1A1B1B1A (lane 2), 7A2B2B1B1C (lane 3)and 7A2B2B1B1D (lane 4)).

FIG. 22 is a Western blot detecting IEfusion protein expression in anexpanded sample that was confirmed to be gus marker-free andwt-MVA-free, and that had a high IEfusion gene copy number.

FIG. 23 is a schematic representation of the mH5-IEfusion-pZWIIA (GUS)construct that is inserted at the Deletion II (del II) insertion site onthe rMVA Viral DNA and the hH5-pp65-pLW51 (GUS) construct that isinserted at the Deletion III (del III) insertion site on the rMVA ViralDNA.

FIG. 24 is a scheme of the MVA genome showing deletions and intergenicregions (IGRs). The MVA deletion III insertion site (del III) for themH5-pp65-pLW51 (GUS) shuttle plasmid is shown by the arrow. The shuttleplasmid has insertion sites for foreign genes (pp65) controlled by anmH5 promoter, a marker gene (gus), homologous flanking sequences forrecombination (FL1, FL2), ampicillin resistance (amp) and direct repeatsfor marker gene removal (DR).

FIG. 25 is a Western blot verifying pp65 incorporation by restrictionenzyme digestion (Asc I and Pme I) followed by DNA sequence analysis.

FIG. 26 is a set of Western blots detecting IEfusion (A) and pp65 (B) infour expansion candidates (14B1C2A3B (lane 1), 14B1C2E4B (lane 2),14B1C2E7C (lane 3), and 14B1C2F1B (lane 4))

FIG. 27 is a set of Western blots detecting IEfusion (A) and pp65 (B) intwo expansion candidates, F8 (lane 1) and 23D5 (lane 2), that were gusmarker-free, parental MVA-free and that had equivalent copy numbers ofIEfusion and pp65.

FIG. 28 is a set of Western blots detecting IEfusion (A) and pp65 (B) inthe selected virus seed, candidate F8.

FIG. 29 is a table representing qPCR data for establishing the IEfusionstandard curve for and the copy number of sample 8B1A1A1A. based on saidstandard curve.

DETAILED DESCRIPTION OF THE INVENTION

rMVA vaccines, rMVA viruses and their antigenic components, methods forproducing the rMVA vaccines and methods of their use are provided. SuchrMVA vaccines comprise immunologically effective amounts of rMVA virusesthat express one or more foreign protein antigens under the control of amodified H5 (mH5) promoter, and methods for their production. In someembodiments, the foreign protein antigens are cytomegalovirus (CMV)antigens as described below. The vaccines described herein comprise animmunologically effective amount of said rMVA viruses that exhibitimmunogenicity and are genetically stable after serial passage. TheserMVA vaccines may be used, for example, as a vaccine to prevent, controlor treat CMV) infection.

In one embodiment, an rMVA vaccine comprising an immunologicallyeffective amount of rMVA virus which is genetically stable after serialpassage and can be produced by genetically engineering MVA viruses toexpress one or more foreign protein antigens under the control of amodified H5 (mH5) promoter. For example, an MVA transfer plasmid vectorcan be constructed first, which plasmid comprises a vaccinia mH5promoter operably linked to a DNA sequence encoding one or moreheterologous foreign protein antigens of interest, wherein theexpression of the DNA sequence is under the control of the mH5 promoter.The plasmid may further contain DNA sequences coding for proteins usedfor screening or selection of rMVA viruses. The DNA coding sequence isin frame with the promoter, i.e., the vaccinia promoter and the DNAcoding sequence (e.g., genes of interest and genes for screening orselection purposes) under the control of the promoter should havecontinuous open reading frames for expression of genes of interest.Next, rMVA viruses are generated by transfecting the plasmid vectorobtained from the first step into wild type MVA virus for homologousrecombination between the transfer plasmid(s) and the MVA backbonevector. See, e.g., FIG. 5A. An rMVA virus expressing the foreign proteinantigen coding sequence can be selected by visible phenotype of the rMVAvirus or by screening methods as further described below. The selectedrMVA viruses are then purified or isolated to form the desired vaccinestock. The Examples below further illustrate more detailed proceduresfor the production of the genetically stable rMVA vaccine.

An “immunologically effective amount” as used herein means an amountthat is both safe to a subject (animal or human) to be immunized andsufficient to improve the immunity of the subject. The immunologicallyeffective amount can vary and can be determined by means of known artthrough routine trials.

In another embodiment, a cytomegalovirus (CMV) vaccine containing animmunologically effective amount of rMVA virus which is geneticallystable after serial passage can be produced by the methods as describedsupra, in which the CMV gene is the gene of interest.

In one aspect of one embodiment, the foreign protein antigens maycomprise one or more CMV antigens. Evidence from studies of murine CMV(MCMV) point to the important role of IE1 antigens for development ofprotective immunity in transplantation models (Reddehase et al. 1987).More recently, homologues of the human CMV pp65 antigen assembled intoviral or plasmid DNA vectors showed evidence of stimulating protectiveimmunity against murine CMV (MCMV), guinea pig CMV (GPCMV), or RhesusCMV (RhCMV) (Morello et al. 2000; Schleiss et al. 2007; Yue et el.2007). Further, an MVA may be developed into a vaccine for deliveringCMV antigens and then clinically evaluated as to which of them exhibitprotective qualities in the context of CMV complications resulting fromtransplant procedures (Song et al. 2007; Wang et al. 2004a; Wang et al.2004b; Wang et al. 2007).

A CMV antigen can be a CMV protein antigen, a fragment of a CMV proteinantigen, a modified CMV protein antigen, a fragment of a modified CMVprotein antigen, a mutated CMV protein antigen or a fusion CMV proteinantigen. Examples of CMV protein antigens and CMV fragments may includepp65, CMV pp150, IE1, IE1 exon 4 (IE1/e4), IE2 exon 5 (IE2/e5),glycoprotein B (gB) and antigenic fragments thereof. Examples ofmodified CMV protein antigens and fragments thereof may be found in U.S.Pat. No. 7,163,685 to Diamond et al. and is incorporated herein byreference in its entirety. Examples of mutated CMV protein antigens maybe found in U.S. Pat. No. 6,835,383 to Zaia et al. and is incorporatedherein by reference in its entirety.

Fusion CMV protein antigens may comprise two or more CMV proteins,modified CMV proteins, mutated CMV proteins or any antigenic fragmentsthereof. In some embodiments, a fusion CMV protein is an IEfusionprotein, comprising a sequence that encoded two or more antigenicportions of Immediate-Early Gene-1 or Immediate-Early Gene-2. In oneembodiment, an IEfusion protein is a fusion of IE1 exon 4 (IE1/e4) andIE2 exon 5 (IE2/e5), IE1/e4-IE2/e5 (IEfusion). Previous studies in CMVvaccine development point to robust immunity in mouse models using anMVA expressing pp65 and IE1 exon4 (Wang et al. 2007). The development ofan IEfusion protein incorporating the adjacent exon5 increases thecoverage of additional human leukocyte antigen (HLA) types by thevaccine, because of the different HLA recognition profiles for both IE1and IE2 genes. Profound sequence differences between the major exons ofboth IE1 and IE2 genes result in a substantial difference in epitopemotifs represented in both genes that is manifested by distinctlydifferent immunologic profile of recognition among individuals whorecognize either gene product. In one embodiment, the use of fusionproteins involves creating an IEfusion protein that comprises exon4 fromIE1 and exon5 from the IE2 gene into a single gene without additionalgenetic material. The IEfusion protein comprises a more completerepresentation of the immediate-early antigens than either proteinalone. Example 2 illustrates the construction of the IEfusion gene, itscloning into a transfer vector (pZWIIA), and generation of rMVA vectorthat expresses the IEfusion gene with or without co-expression of pp65.

In one embodiment, the nucleic acid sequence encoding pp65 has asequence containing nucleotides 3235-4920 of SEQ ID NO: 9 (FIG. 8B). Inanother embodiment, the nucleic acid sequence encoding the IEfusionprotein is SEQ ID NO:11.

To evaluate the IEfusion gene as an immunogen, extensive analysis wasperformed to establish parameters of expression and immunogenicity toqualify it for potential clinical use. Two forms of rMVA were designedto test the IEfusion protein, either as a single antigen or combinedwith pp65. Strong expression of the IEfusion protein as a single-antigenMVA or in combination with pp65 was shown. In either virus, the antigenwas strongly expressed behind the synthetic E/L promoter (pSyn I). Thisdemonstrates that the presence of pp65 did not interfere with IEfusionprotein expression, contrary to earlier reports of interference (Gilbertet al. 1993; Gilbert et al. 1996). This result confirms the robustimmunogenicity of an rMVA vaccine expressing IEfusion with or withoutco-expression of pp65 in mouse models as well as studies in humanperipheral blood mononuclear cells (PBMC). These results are shown inExample 1 below. Immunogenicity analysis of this MVA vaccine is based onmeasuring CMV-specific IFN-γ+ T cell responses, which correlates withcytotoxic function in mouse models and protective immunity in clinicalsituations (Avetisyan et al. 2007; Sinclair et al. 2004). Theimmunogenicity of the recombinant MVA expressing pp65 and IEfusionantigens provides a strategic approach for developing a CMV vaccine fortransplant recipients.

An rMVA that expresses IEfusion with or without co-expression of pp65should expand the diversity of cellular immune responses against CMV tocounter viremia in an immunosuppressed patient. The rationale of thisvaccine virus is to include antigens from CMV that are expressed earlyto disrupt the viral life cycle. The representation of IE-specificimmunity was maximized by including both the IE1 and IE2 antigens. pp65,IE1, followed by IE2, rank among the best recognized antigens in the CD8subset, and with the largest proportion of the T cell memory response toCMV (Sylwester et al. 2005). There is no region of homology greater than5 amino acids between the major exons of both proteins. Individually,both antigens are recognized broadly by almost 70% of the generalpopulation (Sylwester et al. 2005). While few epitopes have been mappedto unique sequence positions of either gene, the divergent sequence ofboth IE1/e4 and IE2/e5 used here predicts an entirely different subsetof HLA binding peptides using publicly available Class I and II motifalgorithms (Peters and Sette 2007). Human subjects that were evaluatedfor recognition of both IE1 and IE2 antigens were found in manyinstances to recognize one or the other but not both. Among the researchsubjects analyzed, 24% recognized IE2 with or without pp65 to theexclusion of IE1. This result strongly suggests that the recognitionelements for both antigens are unique, and by including both of them inthe vaccine, the breadth of individuals with disparate HLA types thatwill recognize and develop an immune response to the vaccine isextended. The fusion of major exons from both antigens achieves the dualgoal of reducing the number of separate inserts and eliminating the needfor a third insert promoter. The advantages of this approach includeplacement of all vaccine antigens in one vector, and diminishing thedose of virus needed to attain sufficient immunity simultaneouslyagainst all of the included antigens.

Humanized transgenic (Tg) mice that do not express murine Class Ialleles (Lemonnier 2002) are available in a variety of forms thatexpress human HLA A2, B7, A11, providing the most direct way to assessHLA recognition of vaccines in a mouse model (Firat et al. 2002). Theprocessing of both rMVAs was first examined utilizing HHD II mice, whichare known to be effective in processing and recognition of poxvirusesspecific for a wide variety of infectious pathogens, including CMV(Daftarian et al. 2005; Gomez et al. 2007; Wang et al. 2004b). Theresults confirm that the IEfusion antigen in MVA is processed andimmunologically recognized throughout both exons, and the fusion jointdoes not impede this process. IFN-γ expression levels were used toassess T cell recognition of CMV antigens expressed from the vaccine,which is shown to have a strong correlation with cytotoxic function inmouse models (Daftarian et al. 2005; Song et al. 2007; Wang et al.2004a). In addition, measurement of IFN-γ production has been reliedupon to assess CMV immunity in CMV-infected healthy individuals(Ghanekar et al. 2001; Sinclair et al. 2004; Sylwester et al. 2005).

To further assess the capacity of the rMVA vaccine to be recognized in avariety of HLA context, B7 mice with a similar C57BL/6 background as theHHDII mice were also immunized with the pp65-IEfusion-MVA andinvestigated for immunogenicity using the same approach as with theHHDII mice. Highly effective recognition of the pp65 antigen was foundas well as a CD8 response to the IE2 antigen using a peptide library.This illustrates that rMVA is processed efficiently by multiple HLAalleles, and provides further support for its utility as a clinicalvaccine strategy. While HLA Class I Tg mice serve a fundamental andirreplaceable role to demonstrate the immunogenicity of the MVAconstructs, they cannot be directly compared with human in vitroclinical results. The in vitro clinical results are best suited to becompared with human research subjects, because mice and human immunerepertoires are not identical. As humans express a diversity of HLAalleles, a multi-antigen vaccine can encompass as many as possible tobroaden the applicability of the vaccine to outbred human populations.While the Tg mice are a valuable tool to evaluate HLA Class I restrictedCD8 T cell responses, they have an intact full complement of murine MHCClass II genes and cannot be directly compared to humans who possess adifferent repertoire of Class II MHC genes and DNA sequences. pp65elicits the strongest CD4 response of the three antigens in both miceand humans. In contrast both IE1 and IE2 do not elicit strong CD4-basedimmunity in both mice and humans (compare FIGS. 11 and 13).

Prior to conducting experiments with rMVA in clinical samples, thecapacity for stimulation of both CD4+ and CD8+ T cells was assessedusing the commercially available pp65 and IE1 library and a newlydesigned IE2 peptide library. Relationships among the T cell populationswere similar to prior results: pp65 promotes a substantial CD4 and CD8response in over 70% of participants, while IE1 and IE2 are recognizedless frequently and mainly in the CD8+ T cell compartment (Khan et al.2002; Khan et al. 2007; Sylwester et al. 2005). This confirms that theIE2 formulation is a reagent of equal potency to the commerciallyavailable pp65 and IE1 peptide libraries to assess memory T cellresponses in the volunteer pool, and should be an effective detectionreagent of memory immune responses to rMVA. Recognition of all threelibraries was evaluated in transplant recipients in all three riskgroups including those with CMV-positive or -negative donors orthemselves being CMV-negative with a CMV-positive donor. This study isunique because no previous evaluation of peptide libraries has beencarried out with HCT recipients using all three antigens simultaneously(Lacey et al. 2006). Patient samples were examined at day 180post-transplant to minimize the effects of myeloablation and incompleteimmune reconstitution on the recognition of the peptide libraries. Theimmune recognition of all three libraries was successful in allpatients, and the relative proportion of T cells that responded to eachlibrary also mirrored what was found in the healthy volunteers.

rMVA expressing the IEfusion antigen with or without the pp65 antigenwas evaluated in PBMC from healthy volunteers to establish theirrecognition properties using a fully human system. The results showedthat the memory T cell expansion stimulated by the rMVA for both theIEfusion and pp65 antigens, followed the proportions found ex vivo forthe same volunteers using the peptide library approach. While there wassubstantial amplification of the relevant T cell populations, thestimulation did not skew the population towards a particular subset orantigen specificity. The data also confirms that the IEfusion protein isprocessed and presented appropriately to stimulate existing T cellpopulations in a manner that maintains the phenotypic distribution asexpected in the ex vivo analysis. This outcome is most favorable towardsusing the rMVA as a vaccine in both CMV positives and negatives, sinceit is preferable to maintain the proportion of T cells that areassociated with an asymptomatic phenotype and hopefully induce that sameproportion in CMV-negative subjects. Stimulation of primary immunity inCMV-naïve mice was successful using in vivo immunization, but not fromclinical samples in the CMV-naïve subject that was investigated. Theconditions of in vitro immunization are insufficient in most cases todrive primary immunity, because the architecture of the lymph node,thymus and dendritic cell systems is missing, so the T cell precursorsshould pre-exist or form in culture. Developing primary immunity to CMVpost-transplant is often delayed in the CMV-naïve recipient or donor inthe case of stem cell transplant, and is thought to be a risk factor forCMV disease (Limaye et al. 2006; Ljungman et al. 2006). Precedent forpoxvirus-based CMV vaccines to stimulate primary immunity wasestablished with a single-antigen pp65-ALVAC used in a clinical trialconducted with CMV-negative healthy volunteers (Berencsi et al. 2001).

The rMVA vaccine described herein overcomes the unreliability of invitro immunization for eliciting primary immunity. It also provides analternative approach to adoptive transfer, which is not feasible whenconsidering a CMV-negative donor for HCT or a CMV-negative recipient ofa CMV infected donor organ (Walter et al. 1995). It is problematic forCMV itself to serve as a stimulator for characterizing memory responses.The concurrent activating and immunosuppressive properties of CMV canconfound interpretation of immunologic methods using it for in vitrostimulation (Manley et al. 2004). In fact, one needs to artificiallyremove the immune-evasion genes from CMV in order to elicit a diverseimmune response that includes the IE antigens, a fact that has beenstressed in the literature (Khan et al. 2005; Manley et al. 2004).Laboratory strains of CMV that are the only practical approach forgrowing the virus to assess recall immunity are plagued with anartificial excessive accumulation of the pp65 protein that interfereswith the recognition of IE proteins which also has been discussed in theliterature and has been the source of controversy in the field (Gilbertet al. 1996; Kern et al. 1999; Wills et al. 1996).

In contrast, it has been shown that MVA vaccines composed of CMV subunitantigens (e.g. pp65, IE1, and gB) can elicit primary immunity inCMV-naïve rhesus macaques, even offering partial protection against achallenge dose of rhesus CMV (Yue et al. 2008). The profile of immuneresponses that are stimulated by MVA are different than what could beelicited using CMV as a viral stimulator in culture methods.Consequently, since rMVA or ALVAC expressing CMV antigens expand T cellpopulations in both CMV-naïve (mice and macaques) and experienced(human) hosts, one application of a CMV vaccine described herein is inthe high-risk CMV-negative transplant recipient for protection againstthe effects of a CMV-infected organ. One example would introduce thepp65-IEfusion-MVA as a vaccine into the CMV-negative recipient as aprecaution several months before transplant (Khanna and Diamond 2006; LaRosa et al. 2007). Another application is to use pp65-IEfusion-MVA as avehicle to expand T cell populations from CMV-positive donors of HCT,and infuse the amplified T cells into a transplant recipient with activeviremia.

The most rigorous evaluation of the processing of the rMVA for T cellresponse is using PBMC from transplant patients. PBMC from HCTrecipients in all three risk categories were evaluated and anequivalently strong recognition of both rMVAs was found. In some cases,it was even more vigorous than in the PBMC of healthy adults. Nointerference with the recognition of the IE antigen by the co-expressedpp65 antigen was found from the same rMVA, which further confirms thatthe recognition of both antigens can take place at the same time andderived from the same vector. Prime-boost strategies utilizingheterologous vaccines, including DNA and viral vectors, suggest improvedimmunogenicity in several pathogen models, including malaria and HIV(Barouch et al. 2003; Gilbert et al. 2006; Goonetilleke et al. 2006;Peters et al. 2007). The ongoing evaluation of a DNA vaccine against CMVsuggests a worthwhile strategy of combining MVA with a plasmid DNAvaccine. The excellent track record of MVA used as a vaccine in theimmunosuppressed population makes it an ideal candidate as a therapeuticin HCT recipients (Cosma et al. 2003; Mayr and Danner 1978; Stittelaaret al. 2001).

The term “genetic stability” as used herein refers to a measure of theresistance to change, with time or serial passage of virus, of the DNAsequence of a gene, the expression level of the gene, or both. Thegenetic stability of the target gene in an rMVA vector is a concern inthe development of a vaccine. A reduction of the genetic stability ofthe target gene may have the effect of reducing the immunogenicity ofthe rMVA vector due to changes in gene sequence or expression level.

Genetic stability of recombinant virus can be measured or assessed bynumerous methods known in the art, e.g., testing foreign proteinexpression levels at each passage by Western blot (WB) or immunostainingvirus plaques and calculating the percentage of foreign proteinproducing foci before and after serial passage (de Haan et al 2005; Timmet al. 2006; Wyatt et al. 2008a; Wyatt et al. 2008b). These methods aretime-consuming and labor intensive. An alternative method to assessgenetic stability is by quantitative PCR (qPCR), which amplifiesisolated MVA genomic DNA and calculates the copy numbers of the insertedgene of interest and MVA vector after each passage. The ratio of thegene of interest copy number versus the MVA backbone vector copy numberis used to determine the genetic stability of the gene or the MVAvaccine carrying the gene. A higher ratio of the gene of interest copynumber to the MVA backbone vector copy number reflects a higher geneticstability, with the highest ratio=1 means approximately 100% geneexpression remains after serial passage. qPCR is more sensitive,high-throughout and provides highly reproducible results relative toother methods, such as Western blot or immunostaining. The method ofqPCR can be performed following well known procedures in the art or themanuals of commercially available qPCR kit, which is also illustrated inExample 2 below. The TaqMan PCR method can also be adapted for stabilitytesting as previously described (Butrapet et al. 2006)

An rMVA vaccine carrying a gene of interest is genetically stable whenthe DNA sequence of the gene and the expression of the gene issubstantially unchanged over the time or serial passage of the vaccine,particularly, after 5 or more passages, more particularly, after 10 ormore passages.

As illustrated in the examples below, with a homogenous initial virusstock of MVA expressing pp65-IE1 or pp65-IEfusion under strong promoterpSyn, 100% (6 of 6 isolates) of individual isolates from passage 1 (P1)had pp65 expression. However, pp65 protein expression levels decreasedsignificantly during serial passage. About 40% (8 of 18 isolates) ofindividual isolates of pSyn-pp65-IE1-MVA had lost pp65 proteinexpression in passage 10. Southern blot assays for pp65 and IE1 geneinsertion demonstrated that non-expressing mutant isolates lost theentire gene expression cassette at the deletion II region ofpSyn-pp65-IE1/e4-MVA (FIG. 1C).

As illustrated in Example 2 below, the deletion II region of the MVA hasbeen studied using restriction endonuclease analysis of MVA genomic DNAand qPCR using a series of primers (SEQ ID NOs: 1-8) that targeted thesurrounding deletion II (del II) region. The pp65 and IE1 geneexpression cassettes including pSyn promoters together with thesurrounding MVA del II region were absent after passages. As shown inExample 2, pSyn-pp65-IEfusion-MVA was serially passaged five times.However, instability was observed after a single passage.pSyn-pp65-IEfusion-MVA was highly unstable, as only 10% of the CMV-pp65and IEfusion gene copies remained after 5 passages. This means that 90%of the original levels of pp65 and IEfusion insert sequences were lostas detected by qPCR. See FIG. 4B. pSyn-pp65-IE1/e4-MVA was slightly morestable, and had a 70% loss of insert gene copies after serial passage.These data are consistent with stability of rMVA being negativelyaffected by the type of insertion because the only difference betweenpSyn-pp65-IE1-MVA and pSyn-pp65-IE1/e4-MVA is the addition of IE2 exon5gene and its fusion to IE1 exon4.

The molecular mechanism for genetic instability of rMVA using pSynpromoter and improved genetic stability using mH5promoter has yet to befully investigated. The cause for the instability of rMVA may be due tohigh protein expression levels, which may be toxic to the cells whichare infected by the rMVA, since the pSyn promoter is optimized to attainhigh levels of transcriptional activity (Chakrabarti et al. 1997; Wyattet al. 2009; Wang et al. 2010). Instability problems have also beenobserved with respect to HIV-Env and the measles virus F proteinexpressed in MVA (Gomez et al. 2007; Stittelaar et al. 2000). In bothcases, toxicity of the expressed protein contributed to geneticinstability of the rMVA expressing them (Wyatt et al. 2009). Further, ithas been reported that rMVA expressing hemagglutininneuraminidase (HN)glycoproteins under control of the vaccinia pSyn promoter replicatepoorly due to toxic levels of the gene product (Wyatt et al. 1996). TherMVA expressing PIV3 F and HN genes under control of pSyn replicatespoorly whereas rMVA expressing both genes under control of mH5 promotercan replicate to high titer in CEF cells due to less expression of PIV3and HN (Wyatt et al. 1996). Genetic stability of rMVA was enhanced byreducing expression levels of HN glycoproteins.

As illustrated by the Examples below, stable expression of foreignprotein antigens, and thereby immunogenicity, of rMVA vaccines afterserial passage can be rendered by expressing the foreign proteinantigens under control of a mH5 promoter. For example, in MVA vectorsexpressing pp65, pp65-IE1/e4, pp65-IE2/e5, or pp65-IEfusion, mH5promoter, which is a weaker promoter than pSyn, directs more stableexpression after serial passage, thereby translates to more potentimmunogenicity, relative to expression and immunogenicity exhibited byMVA vectors expressing the same proteins under pSyn.

Thus, an rMVA virus that expresses one or more foreign protein antigensunder the control of modified H5 (mH5) promoter exhibits both geneticstability and immunogenicity after serial passage (Wang et al. 2010). Inone embodiment, the nucleic acid sequence encoding vaccinia mH5 promoterhas a sequence containing nucleotides 3075-3168 of SEQ ID NO: 9 or3022-3133 of SEQ ID NO: 10 (FIGS. 8A and 8B).

The construction of the rMVA vector can be made by well-knowntechniques. See, e.g., Maniatis et al. Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press (1982). For example, an MVAtransfer plasmid containing IE, IEfusion and pp65 under pSyn or mH5promoters can be constructed. See Examples 1 and 2. The expressingcassettes can be constructed with one promoter directing the expressionof one or more genes of interest or in the form that each gene ofinterest is under control of a separate promoter. The plasmids aretransfected into wild type MVA virus stock to produce rMVA viruses.Serial passages of rMVA viruses are conducted and expression levels ofthe CMV antigens are measured by Western blot or qPCR. Primers for qPCRcan be designed with knowledge of gene information of the desired orinterested foreign protein antigen and genomic information of MVA, e.g.,CMV antigen coding gene MVA DNA sequence information illustrated inFIGS. 8A and 8B. The genetic stability and immunogenicity can beassessed after each passage and the final passage, as illustrated byExample 2 below (qPCR).

In one embodiment, an rMVA vector that expresses IEfusion and pp65 maybe constructed from two MVA transfer plasmids, mH5-IEfusion-pZWIIA (GUS)(FIGS. 7A and 8A; SEQ ID NO:9) and mH5-pp65-pLW51 (GUS) (FIGS. 7B and8B; SEQ ID NO:10).

The mH5-IEfusion-pZWIIA (GUS) plasmid (9.388 kbp) (FIG. 7A) has thefollowing major sequence features that are integrated into MVA: (i) anMVA deletion II flanking region 1 (FL1) (SEQ ID NO:9; nucleotides1-575), (ii) a restriction enzyme site and linker sequence (SEQ ID NO:9;nucleotides 576-599), (iii) an MVA direct repeat (DR) (SEQ ID NO:9;nucleotides 600-884), (iv) a P7.5 vaccinia promoter sequence (SEQ IDNO:9; nucleotides 885-919), (v) a bacterial gus marker gene (GUS) (SEQID NO:9; nucleotides 920-2739), (vi) an MVA direct repeat (DR) (SEQ IDNO:9; nucleotides 2740-3021), (vii) a vaccinia mH5 promoter andrestriction site sequence (SEQ ID NO:9; nucleotides 3022-3133), (viii)an HCMV IEfusion gene (SEQ ID NO:9; nucleotides 3134-5842), (ix) arestriction site sequence (SEQ ID NO:9; nucleotides 5843-5931), (x) anon-bacterial origin pGEM-4Z vector backbone (SEQ ID NO:9; nucleotides5932-6009), and (xi) an MVA deletion II flanking region 2 (FL2) (SEQ IDNO:9; nucleotides 6010-6399). In addition, the transfer plasmid includesthe bacterial plasmid backbone (SEQ ID NO:9; nucleotides 6400-7896 andnucleotides 8758-9388) which corresponds to pGEM-4Z (accession no.X65305) and the bacterial ampicillin resistance gene (SEQ ID NO:9;nucleotides 7897-8757). See FIG. 8A.

The mH5-pp65-pLW51(GUS) plasmid (8.152 kbp) (FIG. 7B) has the followingmajor sequence features that are integrated into MVA: (i) an MVAdeletion II flanking region 1 (FL1) (SEQ ID NO:10; nucleotides 1-652),(ii) an MVA direct repeat (DR) (SEQ ID NO:10; nucleotides 653-933),(iii) a P11 vaccinia promoter sequence (SEQ ID NO:10; nucleotides934-975), (iv) a bacterial gus marker gene (GUS) (SEQ ID NO:10;nucleotides 976-2794), (v) an MVA direct repeat (DR) (SEQ ID NO:10;nucleotides 2795-3074), (vi) a vaccinia mH5 promoter and restrictionsite sequence (SEQ ID NO:10; nucleotides 3075-3168), (vii) a multiplerestriction site sequence (SEQ ID NO:10; nucleotides 3169-3234)(PmeI/SalI/ClaI/HindIII/EcoRI/EcoRV/PstI), (viii) an HCMV pp65 gene (SEQID NO:10; nucleotides 3235-4920), (ix) a restriction site sequence (SEQID NO:10; 4921-4941) (AscI/PstI), and (x) a MVA deletion II flankingregion 2 (FL2) (SEQ ID NO:10; nucleotides 4942-5330). In addition, thetransfer plasmid includes the bacterial plasmid backbone (SEQ ID NO:10;nucleotides 5331-6672 and nucleotides 7534-8152) which corresponds topGEM-4Z (accession no. X65305) and the bacterial ampicillin resistancegene (SEQ ID NO:10; nucleotides 6673-7533). See FIG. 8B.

The process of rMVA vector construction may include various selectionmethods in order to select only those vectors that contain desiredcharacteristics. For example, one embodiment is directed to theconstruction of an rMVA containing IEfusion wherein an IEfusion plasmidis transfected into wild type MVA virus stock. The resulting populationcomprises unsuccessfully transfected MVA and successfully transfectedrMVA that contains IEfusion. An antibody-based screening approach isthen used to screen out the unsuccessfully transfected MVA. Anotherembodiment is directed to the construction of an rMVA containing pp65wherein a pp65 plasmid is transfected into wild type MVA virus stock.The resulting population comprises unsuccessfully transfected MVA andsuccessfully transfected rMVA that contains pp65. An antibody-basedscreening approach is then used to screen out the unsuccessfullytransfected MVA.

A further embodiment is directed to the construction of an rMVAcontaining IEfusion and pp65 wherein a pp65 plasmid is transfected intorMVA that contains IEfusion. The resulting population comprisesunsuccessfully transfected rMVA that contains IEfusion and successfullytransfected rMVA that contains IEfusion and pp65. An antibody-basedscreening approach is then used to screen out the unsuccessfullytransfected rMVA. Construction of an rMVA containing IEfusion and pp65may also be attained wherein an IEfusion plasmid is transfected intorMVA that contains pp65. The resulting population comprisesunsuccessfully transfected rMVA that contains pp65 and successfullytransfected rMVA that contains pp65 and IEfusion. An antibody-basedscreening approach is then used to screen out the unsuccessfullytransfected rMVA.

According to the invention, an rMVA virus can be selected by visiblephenotype, if any. Many recombinant screening methods known in the artor their combinations can also be used for identifying rMVA viruscarrying the rMVA virus vector. For example, by targeting the foreigngene to the thymidine kinase (TK) locus, recombinant viruses can beselected by their TK-negative phenotype in TK-deficient cells.Alternatively, the transfer vector may enable the co-integration of anantibiotic selection marker or a reporter gene allowing color screeningdue to β-galactosidase or β-glucuronidase synthesis. The reversal ofhost range restriction or plaque phenotype can also be used. See, e.g.,Moss B, Genetically engineered poxviruses for recombinant geneexpression, vaccination, and safety, Proc. Natl. Acad. Sci. USA93(21):11341-48 (1996).

The screening methods contemplated by the invention include, but are notlimited to, gene-in (positive selection) and gene-out (negativeselection) methods. rMVA vector construction may include one or morescreening methods that may include all gene-in methods, all gene-outmethods, or any combination thereof.

A gene-in screening method is used to screen rMVA virus to determinewhether a gene of interest is incorporated (by homologous recombination)into the MVA backbone and expressed by the rMVA virus. Examples of thegene-in method include, but are not limited to, antibiotic resistanceselection, colorimetric screening, light or fluorescence screening,nucleic acid testing and immunoscreening.

In one gene-in method, antibiotic resistance selection, the MVA vectorcontains an antibiotic resistance gene such that when MVA virusesreplicate, only those which incorporate the rMVA vector survive in themedium with the corresponding antibiotic. Any antibiotic, mixtures andthe combinations thereof may be used, such as ampicillin, kanamycin,tetracycline, and chloramphenicol and their corresponding resistancegene.

Another gene-in method, colorimetric screening, is a method in which thedetection of a target is indicated by a visible detectable color change,e.g., the detection of the product of a reporter gene genetically onrMVA vector virus binding to a substrate molecule. The strength of thebinding can also be detected by the method and indicated by a colorchange. For example, the rMVA vector can be constructed to include areporter gene such as a lacZ gene or gus gene. The lacZ encodesβ-galactosidase (lacZ). rMVA virus expressing lacZ produceβ-galactosidase which are turned blue by X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside). See Joung et al. Abacterial two-hybrid selection system for studying protein-DNA andprotein-protein interactions, Proc Natl Acad Sci USA, 97(13): 7382-87(2000). Another reporter gene, gus, encodes beta-glucuronidase, which isan enzyme that can transform the host into colored or fluorescentproducts when incubated with some specific colorless or non-fluorescentsubstrates. See Jefferson et al., beta-Glucuronidase from Escherichiacoli as a gene-fusion marker, Proc Natl Acad Sci 83(22): 8447-51 (1986).Thus, in an rMVA virus carrying a cassette expressing both a gene ofinterest and a reporter gene for colorimetric screening, theco-expression of the reporter gene, such as lacZ or gus gene, togetherwith the gene encoding the foreign protein antigen is indicated by acolor change due to the binding of the reporter gene products, such asβ-galactosidase and beta-glucuronidase, with a substrate molecule.

In another gene-in method, fluorescence screening, the MVA vectorincludes a luminescence or fluorescence gene such that the co-expressionof the luminescence or fluorescence gene together with the gene encodingthe foreign protein antigen is indicated by illumination of light orfluorescence, which is visible by eye or can be detected by aninstrument, such as a fluorescence microscope. One example ofluminescent molecule is luciferase. Light is emitted when luciferaseacts on the appropriate luciferin substrate. Examples of fluorescentmolecule include, but are not limited to green fluorescent protein(GFP), blue fluorescent protein (BFP), red fluorescent protein (RFP),cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP, suchas the Venus™ fluorescent marker, a commercially available YFP variant,used the Examples below). For example, fluorescence gene constructed inthe same open reading frame as the gene coding for the foreign gene inan rMVA vector, the expression of the genes causes the transformed MVAvirus (rMVA virus) plaques to fluoresce. Further details of how tochoose a fluorescent protein can be determined by means of known artthrough routine trials. See Shaner et al., A guide to choosingfluorescent proteins, Nat Methods 2(12): 905-9 (2005).

Nucleic acid testing is another gene-in method used to detect orsequence the nucleic acid molecule of the rMVA vector for a desiredsequence. PCR may be used to detect the desired sequence, e.g., asequence unique in rMVA vector but not in wild type MVA genome. Asillustrated in Example 2, qPCR methods can be used to assess copynumbers of MVA vector and genes of interest after each passage. Primersfor qPCR can be designed with knowledge of gene information of thedesired or interested foreign protein antigen (e.g., cytomegalovirus(CMV) antigen coding DNA sequence information illustrated in FIGS. 8Aand 8B) and genomic information of MVA. Other nucleic acid testingmethods include sequencing the MVA vector DNA to determine whether thecassette expressing the gene of interest is incorporated into the MVAvector backbone sequence via homologous recombination.

Immunoscreening is another gene-in method for detecting the expressionof a gene of interest in an rMVA vector via immunologic reaction. Forexample, the expression of interested or desired foreign proteinantigen, e.g., a cytomegalovirus (CMV) antigen, can be detected by anantibody to the desired foreign protein antigen. See Akoolo et al.,Evaluation of the recognition of Theileria parva vaccine candidateantigens by cytotoxic T lymphocytes from Zebu cattle, Vet ImmunolImmunopathol 121(3-4):216-21 (2008).

Gene-in screening methods are useful in many situations. However, when arecombinant gene or protein is used in the development of gene therapyand vaccination methods, gene-out screening methods may be desired ornecessary to ensure that a particular gene is not expressed in vivo. Agene-out screening method is used to screen rMVA virus to determinewhether a gene of interest is not incorporated, excised or deleted inthe rMVA virus. Such methods may include the use of inserted genes toprovide a “suicide” or “fail-safe” trait that would permit thedestruction of gene-modified cells if they would result in harm to thehost. Other methods may include the removal of a gene used in a gene-inscreening method. For example, an rMVA virus may contain a gus-selectionmarker gene used for isolation of successfully transfected rMVA based onblue color selection in the presence of β-glucoronidase substrate(gene-in). To ensure the isolated rMVA virus that is to be used as avaccine does not contain an unnecessary and potentially allergenicbacterial protein, the gus-selection marker gene is flanked by twodirect repeat sequences to facilitate gus gene removal by intragenomicrecombination (gene-out). See FIG. 9. Other examples of gene-out methodsinclude, but are not limited to toxic prodrug, flanking inverted repeatsand cre-lox system.

A further embodiment is a method for producing a genetically stable rMVAvaccine by genetically engineering MVA viruses to express foreignprotein antigens under the control of a mH5 promoter. As describedsupra, first, an MVA transfer plasmid vector can be constructed, whichplasmid comprises a vaccinia mH5 promoter operably linked to a DNAsequence of interest encoding one or more heterologous foreign proteinantigens, wherein the expression of the DNA sequence is under thecontrol of the mH5 promoter. The plasmid may further contain DNAsequences coding for proteins used for screening or selection of rMVAviruses. The DNA coding sequence is in frame with the promoter, i.e.,the vaccinia promoter and the DNA coding sequence (e.g., genes ofinterest and genes for screening or selection purposes) under thecontrol of the promoter have continuous open reading frames forexpression of genes of interest. Next, rMVA viruses are generated bytransfecting the plasmid vector obtained from the first step into wildtype MVA virus for homologous recombination between the transferplasmid(s) and the MVA backbone vector. See, e.g., FIG. 5A. An rMVAvirus expressing the foreign protein antigen coding sequence can beselected by visible phenotype of the rMVA virus or by screening methodsas further described below. The selected rMVA viruses are then purifiedor isolated to form the desired vaccine stock. The Examples belowfurther illustrate more detailed procedures for the production of thegenetically stable rMVA vaccine. The rMVA vaccine obtained from themethod exhibits genetic stability and maintains immunogenicity afterserial passage, for example, after at least 5 or after at least 10passages.

A further embodiment is a method for producing a genetically stablevaccine as described supra, wherein the foreign protein antigen is a CMVantigen.

Genetic stability of expression and immunogenicity after each passageand the final passage can be assessed as illustrated by Example 2.

Another embodiment is a method for the prevention or treatment ofinfections or cancer in a mammal subject by administering to the subjecta genetically stable rMVA vaccine, wherein the rMVA vaccine contains aforeign protein antigen under control of a mH5 promoter. Anotherembodiment is a method for the prevention or treatment of infections orcancer in a mammal subject by administering to the subject a geneticallystable CMV rMVA vaccine, wherein the rMVA vaccine contains one or moreCMV antigens under control of a mH5 promoter. The subject is a human oranimal subject, for example, a mammal subject or a human subject.

Having described the invention with reference to the embodiments andillustrative examples, those in the art may appreciate modifications tothe invention as described and illustrated that do not depart from thespirit and scope of the invention as disclosed in the specification. Theexamples are set forth to aid in understanding the invention but are notintended to, and should not be construed to, limit its scope in any way.The examples do not include detailed descriptions of conventionalmethods. Such methods are well known to those of ordinary skill in theart and are described in numerous publications. All references mentionedabove and below in the specification are incorporated by reference intheir entirety, as if fully set forth herein.

EXAMPLE 1 CMV Antigens Stimulate Immunity in an rMVA Vaccine Vector

Materials and Methods.

Human Patient Specimens.

The study protocols were approved by the institutional review board atCity of Hope Medical Center (COH), and specimens and data were obtainedprospectively after informed consent was obtained from subjects. PBMCfrom healthy donors and 9 HCT recipients at 180 days post-transplantwere collected at COH and cryopreserved by standard methods (Maecker etal. 2005). HLA typing was performed at the COH HLA Laboratory bystandard SSOP methods. Haplotypes for both Class I and II alleles weredetermined (data not shown). No intentional bias occurred in selectionof HLA types to be included in this study, as all patients or volunteerswere randomly chosen. The CMV serostatus of these subjects wasdetermined as previously described by using latex agglutination(CMV-SCAN; Becton Dickinson) (La Rosa et al. 2007).

Mouse Strains and Conditions of Immunization.

HHD II (Pascolo et al. 1997) and HLA B*0702 (Rohrlich et al. 2003) Tgmice were bred and maintained under pathogen-free conditions in theAALAC-approved animal care facility at COH. Eight to ten-week old groupsof Tg mice (Avetisyan et al. 2007; Barouch et al. 2003) were immunizedi.p. with different rMVA constructs (as described in the figurelegends).

Synthetic Peptides and Monoclonal Antibodies.

HLA-A*0201 pp65₄₉₅₋₅₀₃ (Diamond et al. 1997), HLA B*0702 pp65₂₆₅₋₂₇₅(Longmate et al. 2001) and HLA-A*02 IE1316-324 (Khan et al. 2005)restricted CMV peptides were used as previously described (La Rosa etal. 2001). Overlapping 15-AA peptides (PepMix™) spanning full length CMVpp65 and IE1 proteins were purchased from JPT Peptide Technologies GmbH(Berlin, Germany). Splenic murine cell suspensions were evaluated inflow cytometry using the following antibodies: CD8-FITC (Clone Ly-2),CD4-PE (Clone L3T4), and IFN-γ-APC (Clone XMG1.2). Antibodies used forflow cytometry of clinical specimens include antihuman CD8-PE (CloneRPA-T8), CD4-FITC (Clone RPA-T4) and IFN-γ-APC (Clone B27), purchasedfrom BD-Pharmingen.

Construction of Synthetic IE2 Peptide Library.

The 580 amino acid primary sequence of the IE2 protein [Swiss Prot#P19893] was divided into 15mer stretches that overlap successivepeptides by 11 amino acids using an online program which excludesimpermissible amino acids at the amino (Q) and carboxyl (GPEDQNTSC; SEQID NO:17) terminus of each 15mer peptide based on syntheticconsiderations. 133 peptides were predicted using the algorithm with anaverage length of 15 AA, but tolerating up to 5-AA length variance toeliminate forbidden terminal amino acids. A total of 123 peptides weresynthesized with a Symphony™ peptide synthesizer (Protein Technologies,Inc., Tucson, Ariz.) using standard 9-fluorenylmethoxy carbonylprotocols and purified by high performance liquid chromatography(Agilent Technologies 1200 series) (Daftarian et al. 2005). The mass ofeach peptide was confirmed by matrix assisted laser descriptionionization time of flight analysis using a Kompact Probe™ massspectrometer (Shimadzu Corp., Chestnut Ridge, N.Y.). The library wassub-divided into ˜20 peptides/pool, and subsequently combined into onesuper-pool containing all the component peptides. Several peptides wereimpractical to synthesize, and they did not enter into the poolincluding a 32-AA stretch between 251 and 282 of IE2 sequence. Allpeptides were individually solubilized in 30% acetonitrile/water, except6 with pKs N8, which were dissolved in 0.1% ammonium bicarbonate in 30%acetonitrile/water. The solubilized peptides, sub-divided into sixpools, were further combined into a single super-pool, with each peptideat a concentration of 200 μg/ml, dissolved in 50% DMSO/water, and keptat −80° C. in single use aliquots. The single use aliquots were dilutedinto cell culture medium at a final concentration of 1 μg/ml/peptide forall cellular immunology assays based on previous titration studies.

Construction of Recombination pZWIIA Plasmids for Derivation of rMVA.

The MVA transfer plasmid named pZWIIA with dual vaccinia syntheticpromoters (pSyn I and II) was constructed to facilitate the derivationof bacterial marker gene-free rMVA (Wang et al. 2007). To construct theIEfusion gene, the following primers were designed with syntheticrestriction enzyme sites shown as underlined:

(SEQ ID NO: 12)Primer a: 5′AGCTTTGTTTAAACGCCACCACCATGGTCAAACAGATTAAGGTTCG3′;(SEQ ID NO: 13) Primer b: 5′GGCATGATTGACAGCCTGGGCGAGGATGTCACCCTGGTCAGCCTTGCTTCTAGTCACCAT3′; (SEQ ID NO: 14)Primer c: 5′TGTTAGCGTGGGCCCGGTGCTACTGGAATCGATACCGGCATGATTGACAGCCTGGGCGAGGATGTCACC 3′; (SEQ ID NO: 15)Primer d: 5′TAGCACCGGGCCCACGCTAACAACCCAC 3′; and (SEQ ID NO: 16)Primer e: 5′ TTGGCGCGCCTTTATTTTACTGAGACTTGTTCCTCAGGT3′.

Primers a and b were used to amplify IE1/e4, and after gel purification,the IE1/e4 PCR product was amplified again using Primers a and c anddigested with Pme I and Apa I. Primer b overlaps the junction betweenIE/e4 and IE2/e5 without adding any non-CMV genomic sequence. Primers c(G to C) and d (C to G) contain a single nucleotide change that createsan Apa I site, but does not alter the amino acid sequence. The resultingfragment was cloned into pNEB193 to yield IE1/e4-pNEB193. IE2/e5 wasamplified using Primers d and e and PCR products were digested with ApaI and Asc I and cloned into IE1/e4-pNEB193 to yield IE1-IE2-pNEB193.IE1-IE2 fusion gene (IEfusion) was excised from IE1-IE2-pNEB193 with PmeI and Asc I restriction enzymes and cloned into pZWIIA behind vacciniapSyn I promoter (Chakrabarti et al. 1997) to yield IEfusion-pZWIIA (FIG.9C). To construct pp65-IEfusion-pZWIIA, the 1.7 kb CMV pp65 gene was PCRamplified from an existing plasmid and cloned into Nhe I and Asc I sitesof pZWIIA behind vaccinia pSyn II promoter (Wyatt et al. 2004) to yieldpp65-IEfusion-pZWIIA (FIG. 9C). In neither MVA was pp65 fused to theIEfusion gene. MVA transfer plasmids were verified by restriction enzymedigestion and DNA sequencing.

Generation of IEfusion-MVA and pp65-IEfusion-MVA.

pp65-IEfusion-MVA was generated in CEF cells via homologousrecombination by a transfection/infection method as described previously(Wang et al. 2004a; Wang et al. 2007). pp65-IEfusion-MVA was isolatedbased on blue color selection by nine consecutive rounds of plaquepurification on CEF cells in the presence of β-glucoronidase substrate(X-glcA). IEfusion-MVA was generated similarly to pp65-IEfusion-MVA. Thegus selection marker gene in rMVA virus was screened out using alimiting dilution method as described previously (Wang et al. 2008). WtMVA-free and color marker gene-free recombinant IEfusion-MVA andpp65-IEfusion-MVA virus were expanded and purified by 36% sucrosedensity ultracentrifugation, resuspended in PBS containing 7.5% lactose,retested for protein expression by Western blot (WB), aliquoted, andfrozen at −80° C.

Western Blot Detection of Protein Expression.

The pp65 and IEfusion protein expression levels, measured as separateproteins with distinct molecular weights from IEfusion-MVA andpp65-IEfusion-MVA-infected cells was determined by WB using an enhancedchemiluminescence-based ECL Plus™ detection kit (Amersham PharmaciaBiotech, Buckinghamshire, United Kingdom). Cell lysates were separatedby SDS-PAGE on 10% gels. After electro-transfer of proteins from the gelonto PVDF membranes (Bio-Rad, Hercules, Calif.), the membranes wereincubated first with purified mAb 28-103 (Britt et al. 1985) to detectpp65 as a separate protein, or mAb p63-27 (Plachter et al. 1993) todetect the IE1 or IEfusion protein, followed with HRP-labeled goatanti-mouse Ab according to the manufacturer's instructions.

Ex Vivo and In Vitro Stimulation Conditions for Human PBMC.

Cryopreserved PBMC were rapidly thawed and immediately cultured in 15-mlFalcon tubes at a density of 1 million/ml in RPMI 1640 medium(Invitrogen) supplemented with 10% FCS (Omega Scientific Inc, Tarzana,Calif.) and containing either pp65 PepMix™, IE1 PepMix™ or IE2 peptidelibrary (at a final concentration of 1 μg/ml of the individual peptides)at 37° C. in a 5% CO2-gassed incubator. After 1 h in culture, brefeldinA (GolgiPlug; Becton Dickinson Biosciences) was added, and incubationcontinued for an additional 11 h under the same conditions.

IVS using rMVA was modified from a published method (La Rosa et al.2001). Briefly, cryopreserved PBMCs were rapidly thawed and immediatelydispensed in a 12-well plate at a concentration of 2×106 cells/ml inRPMI 1640 medium (2.5 ml/well) supplemented with 10% human AB serum (COHBlood Bank) and were incubated with 5 μg/ml of both CpG-A ODN 2216 andCpG-B ODN 2006 (TriLink BioTechnologies, San Diego, Calif., USA). After3 days, ODN-treated PBMCs were infected with rMVA expressing pp65 andIEfusion antigens (pp65-IEfusion-MVA), or rMVAexpressing pp65 and IE1exon4 (pp65-IE1-MVA), or rMVAexpressing only IEfusion antigen(IEfusion-MVA) at a multiplicity of infection of 5, for 6 h in RPMI 1640medium with reduced (2%) human AB serum. Infected PBMCs wereγ-irradiated (2500 rad) and used as APC. APC were plated in a 24-wellplate (1.5×106/well), co-incubated with autologous PBMC (3×106/well), ina final volume consisting of 2 ml/well RPMI 1640 medium with 10% humanAB serum and human rIL-2 (NIH AIDS Research and Reference ReagentProgram, 10 units/ml). Every 2 days, 50% of the culture medium wasremoved, and replaced with fresh medium containing rIL2. Cells wereincubated for 8 days and split into additional wells when necessary. Atday 8, cells were collected and washed with medium without rIL-2 andtransferred into 15 ml Falcon tubes. The same stimulation conditions forintracellular cytokine (ICC) assays performed on ex vivo PBMC were usedfor PBMC after IVS.

Intracellular Cytokine Staining of Human PBMC.

After 12 hours of incubation, PBMC were harvested, washed, labeled withPE-conjugated anti-CD8 and FITC-conjugated anti-CD4 antibodies, fixed,and permeabilized (Cytofix-Cytoperm; Becton Dickinson Biosciences)before they were labeled with APC-conjugated antibody to IFN-γ. Thestained cells were analyzed on a FACSCanto™ (BD Immunocytometry Systems,San Jose, Calif.), and data were analyzed using FCS Express (version3.0; DeNovo Software). 0.5×106 events were acquired for each sample.Lymphocytes were initially gated using forward versus side scatter, thenCD4+ and CD8+ lymphocytes cells were gated separately. The number ofIFN-γ expressing cells is shown as a percentage of the CD8+ or CD4+lymphocyte population.

In Vitro Stimulation of Mouse Splenocytes and Detection of CellularResponses.

Three weeks after immunization, spleens were aseptically removed andsplenocytes from individual or pooled mice were stimulated in vitro(IVS) for 1 week with syngeneic LPS blasts as APC, loaded either withthe relevant CMV-CTL epitope or CMV-peptide library (La Rosa et al.2001; La Rosa et al. 2007). The immunological activity of the stimulatedmurine cultures was determined after assessing the levels of IFN-γ CD4+or IFN-γ CD8+ T cells by ICC staining (La Rosa et al. 2001; La Rosa etal. 2007). For CD4, CD8, and IFN-γ labeling, APC-conjugated antibody toIFN-γ, PE-conjugated CD4, and FITC-conjugated CD8 were used (BD, SanJose, Calif.). Flow cytometric acquisition was performed on a FACSCanto™(BD Immunocytometry Systems). Between 0.80 and 1.0×106 events wereacquired for each sample. FACS analysis was performed using FCS Expressversion 2 software (De Novo, Ontario, Canada). The number ofdouble-positive cells is expressed as a percentage of the CD8+ T cellpopulation.

Statistical Analysis Methods.

Ex vivo IFN-γ production versus post-IVS with rMVA by PBMC against pp65,IE1 and IE2 peptide libraries were compared using Friedman's test with 2degrees of freedom, followed by Wilcoxon's rank-sum test for pairwisecomparisons. Comparison of paired data before and after IVS with rMVAwas performed using the Student T-test.

Construction of the IE1/e4-IE2/e5 Fusion (IEfusion) Gene, Cloning intoTransfer Vector pZWIIA and Generation of Recombinant MVA.

IE1, also known as UL123 is composed of four adjacent exons interspersedwith 3 introns. The adjacent UL122 (IE2) gene is composed of the sameinitial 3 exons UL 123 but also contains a unique adjacent exon5 as aresult of alternate splicing (FIG. 9A) (White et al. 2007). Toapproximate the genetic architecture in the CMV genome and to reduce thenumber of independent transcription units to be inserted into MVA, exon4(e4) from IE1 and exon5 (e5) from IE2 were joined as shown in FIG. 9B.Genomic copies of e4 and e5 were amplified from CMV strain AD169 viralDNA, and primers were developed that made use of a newly createdrestriction site in IE2/e5 that was introduced into the IE1/e4 fragmentby PCR methods. The independent exons with overlapping sequence werejoined at the newly created Apa I site to create the fusion gene withoutintroduction of new protein sequence. Exons 2-3 were omitted becausethey contain transcriptional activation domains whose activity mightcause unexpected and undesirable gene activation events. (Gyulai et al.2000; Johnson et al. 1999).

The fusion gene was cloned into pZWIIA using unique restriction enzymesthat were added by PCR to the ends of each exon (FIG. 9C). Versions ofthe transfer plasmid pZWIIA with UL83 (pp65) were also constructed (FIG.9C). pZWIIA encodes two direct repeats flanking the bacterial markergene (such as glucoronidase or gus) that facilitates their removalthrough stochastic recombination as earlier described (Wang et al.2004). Both versions of pZWIIA were used in combination with wild typeMVA to generate rMVA expressing the IEfusion gene alone or co-expressedwith pp65 (FIG. 9D). The pp65 gene was kept separate from the IEfusiongene in the MVA shown in FIG. 9D. Each rMVA underwent ˜8 rounds ofpurification, and was verified to be absent of parental wild type MVA(wtMVA) using PCR methods (Wang et al. 2007).

The artificial joint between IE1/e4 and IE2/e5 was tested as to whetherit would allow continuous translation of the predicted full lengthprotein product, by infecting chicken embryo fibroblast cells (CEF) withwtMVA and simultaneous transfection with pZWIIA containing the IEfusiongene. The results show a 125 kDa protein band composed solely of theIE1/e4 and IE2/e5 exons, detected using an IE1/e4-specific mAb that alsodetected the expected 60 kDa band after infection of CEF with IE1/e4-MVA(Wang et al. 2007). Virus plaques expressing the IEfusion gene with andwithout separately co-expressed pp65 were amplified, and titered viruseswere used to make lysates that were separated using SDS-PAGE, followedby WB analysis using antibodies to detect pp65 (FIG. 10A) and IEfusionproteins (FIG. 10B). The results confirm that the IEfusion protein canbe highly expressed alone or in combination with pp65 (FIG. 10).

Immunogenicity of rMVA that Expresses IEfusion alone or in Combinationwith pp65.

In Transgenic HLA A2 Mice.

To establish whether rMVA would elicit primary immunity in a CMV naivehost, experiments were performed in transgenic (Tg) mice naive to allantigens expressed from the rMVA. HHDII mice which are Tg for the HLA A2gene and focus presentation on the human MHC were immunized with theIEfusion-MVA or pp65-IEfusion-MVA for three weeks (Pascolo et al. 1997).Spleens were processed and in vitro stimulation (IVS) was set up for aperiod of 7 days followed by intracellular cytokine assay (ICC) todetect IFN-γ expression. To evaluate the HLA A2-restricted CD8+ T cellresponse, immunodominant HLA A2-restricted pp65 and IE1 CTL epitopeswere used, as well as the IE2 peptide library, as no HLA A2-restrictedIE2 epitopes have yet been defined (Wang et al. 2004b; Wang et al.2007). To measure MHC Class II CD4+ T cell responses, peptide librariesspecific for the pp65, IE1, and IE2 antigens were used both during theIVS and ICC stimulations.

The results presented in FIG. 11 demonstrate robust immunogenicity ofthe rMVA after infection in the HHDII mouse. Levels of specific IFN-γproduced by CD8+ T cells were significantly higher than for CD4+ T cellsfor all 3 CMV antigens. In contrast, both a robust CD4+ and CD8+ T cellresponse was found for pp65 (FIG. 11). Likewise, there was substantialrecognition of the IE1/e4 portion of the IEfusion protein demonstratedby a potent CD8+ T cell response using the IE1 peptide library (FIG.11). Finally there was a good CD8+ T cell response to the IE2 library,and a lesser response by CD4+ T cells (FIG. 11). The immunogenicity ofthe IEfusion protein was not dependent on the presence of the pp65antigen by immunizing HHDII mice with an MVA that included the IEfusionprotein without coexpression of pp65. The HHDII mice responded similarlyto the IE2 library, and also appropriately responded to the HLA-A2restricted epitope of IE1 in a robust manner. These experiments confirmthe strong immunogenicity of the IEfusion protein, and also verify thatthe immunogenicity of the IE1 portion of the molecule is not disruptedwhen the IE2 portion is fused to it. The immunogenicity of the IE1portion compares favorably to constructs in which IE1/e4 is expressed asa single exon without fusion (Wang et al. 2004b; Wang et al. 2007).

Immunization of Tg HLA B7 Kb^(ko)/Db^(ko) Mice with pp65-IEfusion-MVA.

The success of the immunogenicity trial in HHDII mice led to aninvestigation of a Tg model expressing a different HLA allele togeneralize the scope of immunogenicity of the rMVA in different HLAbackgrounds. B7 mice are deficient in both Kb and Db murine genes, andmainly process Class I antigens using the Tg MHC molecule, HLA B*0702(Rohrlich et al. 2003). Immunization conditions were similar as wedescribed for HHDII mice, and after three weeks, mice splenocytes werestimulated during both IVS and ICC procedures with HLA B*0702pp65₂₆₅₋₂₇₅ epitope to evaluate the Tg CD8+ T cell response. In B7 mice,the recognition of HLA B*0702 IE1 epitopes is minimal, thus the IE1peptide library was used to measure the Tg CD8+ T cell response (FIG.12). Peptide libraries specific for the pp65, IE1, and IE2 antigens werealso used to evaluate the MHC Class II responses. Similar to thefindings in HHDII mice, higher levels of CD8+ and lower levels of CD4+ Tcell responses were elicited against all 3 CMV antigens (FIG. 12). Thisdemonstrates that both the pp65 and IEfusion genes are functional andimmunologically recognized in the Tg HLA B7 mouse model.

Ex Vivo Response to CMV0pp65, IE1 and IE2 Peptide Libraries in HealthyVolunteers and Stem Cell Transplant (HCT) Recipients.

To gauge the strength of the rMVA to stimulate CMV-specific T cells fromPBMC of CMV positives, ex vivo recognition of the three peptidelibraries (pp65, IE1, IE2) corresponding to the cognate expressedproteins in rMVA was examined. Data was taken from 22 CMV-positive (FIG.13) and 8 CMV-negative healthy adult volunteers and classifiedindividuals as a responder if they had antigen-specific T cellfrequencies of greater magnitude than levels found in CMV negatives,which averaged 0.05% of CD8+ and 0.05% of CD4+ T cells for each of the 3peptide libraries. The number of individuals classified as responderswas highest for pp65 in both the CD8 (16/22) and CD4 (10/22) subsets,and there were lower numbers of responders (9/22) for both the IE1 andIE2 library in CD8, but far fewer (3/22) in the CD4 subset. The numberof individuals responding to the 3 peptide libraries is qualitativelysimilar to the only other comparable dataset (Sylwester et al. 2005).The CMV-specific CD8+ and CD4+ T cell frequencies for each of the 22 CMVpositives were calculated, and roughly equivalent responses to all 3libraries in the CD8+ T cell subset were found (FIG. 13A). In contrast,there was a dichotomy of response in the CD4+ T cell subset such thatpp65 responses had a 3-fold higher average than IE responders, which isin line with previous findings (Sylwester et al. 2005). In summary, theT cell responses in the chosen group of CMV positive individuals confirmthe reliability and the legitimacy of using results from a healthyvolunteer group as a benchmark for comparisons with less wellcharacterized HCT patients.

Next, the immune response in HCT recipients was investigated in relationto all three peptide libraries in three combinations of donor (D) andrecipient (R) pairs with increasing risk for complications of CMVinfection (D+/R+, D+/R− and D−/R+) at 180 days post-transplant (FIG.13B). All 9 recipients that we chose were part of a study of naturaldevelopment of immunity to CMV and were known responders to CMV antigens(Gallez-Hawkins et al. 2005; Lacey et al. 2006). All 9 patientsresponded to the 3 peptide libraries by producing a CD8+ T cell responseof similar magnitude to healthy adults with chronic CMV infection (FIG.13A). Similar to the results for healthy volunteers, the pp65 librarystimulated a strong response in both the CD4+ and CD8+ T cell subset,while the IE1 and IE2 libraries were most effective for stimulating aCD8+ T cell response (FIG. 13B). The low level of CD4+ T cell responseto both the IE1 and IE2 libraries consistent with previous reports andthe current results in healthy volunteers. These observations indicatethat both the magnitude and quality of the T cell response to the pp65,IE1, and IE2 antigens are similar in recovering HCT recipients as it isin healthy CMV-positive volunteers.

IEfusion-MVA Stimulates CMV-Specific T Cells in Human PBMC.

The immunogenicity of the IEfusion protein as a single immunogen orco-expressed with pp65 in rMVA was examined. Autologous antigenpresenting cells (APC) were matured to be optimally receptive to MVAinfection and antigen presentation by the use of a CpG DNA cocktail (LaRosa et al. 2006). Following three days of maturation, APC were infectedwith rMVA containing the IEfusion gene or rMVA containing both theIEfusion and pp65 genes, followed by irradiation to inactivate the APCfor proliferation. IEfusion-MVA in PBMC from three CMV-positive healthydonors and one CMV-negative donor was then examined. First, ex vivorecognition of either the IE1 or IE2 peptide libraries was conducted asa comparison to the MVA IVS study (FIG. 14A). The average increase wasquite substantial after IVS with IEfusion-MVA (nearing 5-fold) in eachof the three CMV-positive individuals evaluated in either the CD4+ orCD8+ subset as detected with the IE1 or IE2 peptide libraries (FIG.14A). In contrast, there was no evidence for ex vivo recognition ofpeptide libraries in the CMV-negative individual, nor was there anysignificant stimulation of either IE-specific T cell population. Noevidence of pp65-specific stimulation beyond ex vivo levels was found inCMV positives or negatives, because the rMVA did not express pp65.

The immunogenicity of pp65-IEfusion-MVA was assessed by comparison to exvivo measurements of the autologous PBMC populations using all threepeptide libraries (FIG. 14B). In all individuals examined, there wasbrisk stimulation of antigen-specific T cell populations that oftenexceeded levels found with IEfusion-MVA (FIG. 14A). In the case of theCD8+ T cell subset, IVS caused substantial increase in all threeantigen-specific T cell populations. The ex vivo level of the CD4+subset recognizing pp65 was far greater than for the IE antigens, whichwas also reflected in the amplified frequencies after IVS with rMVA. Thesame CMV-negative healthy donor that was investigated with IEfusion-MVA,had no evidence of pp65 or IEfusion-specific immunity after in vitroimmunization with pp65-IEfusion-MVA. Results from both vaccine virusesestablish that rMVA stimulation does not substantially alter therelationship of the T cell subset proportion measured ex vivo for allthree antigens; it amplifies ex vivo levels to a higher level after IVS(FIGS. 14A and B). As a further control for specificity of CMV antigenrecognition, in vitro immunization of PBMC from 3 healthy donors wasinvestigated as shown in FIG. 14B with an MVA only expressing the gusgene (gus-MVA) that was constructed using different transfer vectors anddescribed in a previous report (Wang et al. 2004a). There was noincremental increase in CMV-specific recognition of all 3 peptidelibraries greater than what was measured ex vivo.

rMVA Stimulates CMV-Specific Effectors in PBMC from TransplantRecipients.

Next, the capability of the three-antigen rMVA to stimulate memoryresponses in PBMC from HCT recipients was evaluated. Two examples werechosen from three different risk categories of patients that were alsoexamined ex vivo: O+/R+, O−/R+ and O+/R− (FIG. 13B). Results of the IVSwith MVA are shown side-by side with the ex vivo response to demonstratethe magnitude of the stimulation of CMV-specific T cell responses in allpatient risk groups (FIG. 15). The CD8+ was more substantial than theCD4+ T cell stimulation which reflected the ex vivo profile, which showssubstantial over-representation of CD8 versus CD4 responses (FIG. 15).The levels of rMVA amplification of CMVspecific T cells in many casesexceed those found in healthy volunteers (FIGS. 14 and 15). This isevident in both the CD4 and CD8 T cell populations, and is observed inall three patient groups with different combinations of CMV serostatus.While not all antigens were equally stimulated in all patients, themajority of measurements demonstrate a substantial amplification from exvivo levels in both the CD4+ and CD8+ T cell population. The specificityof the immune responses to CMV antigens was confirmed by including anadditional in vitro immunization culture using gus-MVA, from two of thesix HCT patients that had sufficient PMBC to conduct this additionalcontrol. There was no evidence of CMV-specific immune stimulation,beyond what was measured ex vivo from both individuals (FIG. 15).

EXAMPLE 2 Increased Stability of CMV Antigens Under Control of mH5Promoter

Materials and Methods

Cells, Virus, Peptides, and Mice.

Primary CEF cells prepared from specific pathogen-free chicken eggs werepurchased from Charles River SPAFAS (North Franklin, Conn., USA). BHK-21cells (ATCC CCL-10) were purchased from American Type Cell Collection(Manassas, Va., USA) and maintained in minimal essential medium (MEM)supplemented with 10% fetal calf serum in a 37° C. incubator containing5% CO2.

Wild type (wt) MVA virus stock, pLW51 and pSC11 transfer plasmids werekindly provided by Dr. Bernard Moss (Laboratory of Viral Diseases,NIAID, NIH). rMVA expressing CMV pp65 alone (pSyn-pp65-MVA) or togetherwith IE1/e4 under control of pSyn promoter (pSyn-pp65-IE1/e4-MVA) weregenerated as previously described [Wang et al. 2007]. rMVA expressingCMV pp65, IEfusion protein (IE1/e4 and IE2/e5) under control of pSynpromoter (pSyn-pp65-IEfusion-MVA) were also developed using a homologousrecombination method (Wang et al. 2008).

Construction of MVA Transfer Plasmids and Viruses Containing mH5Promoters.

pZWIIA transfer vector containing two pSyn promoters was constructed aspreviously described (Wang et al. 2007). Additional MVA transferplasmids were constructed after replacement of pSyn with the mH5promoter. The two pSyn promoters in pZWIIA were replaced with one mH5promoter. Briefly, a 228 bp DNA fragment including the 70 bp mH5promoter sequences and multiple cloning sites was synthesized (Genebankaccession # FJ386852) and cloned into pZERO-2 (Integrated DNATechnologies, Coralville, Iowa). This 228 bp DNA fragment was excisedwith Xho I and Not I, gel purified and cloned into pZWIIA to yieldmH5-pZWIIA. The mH5-pZWIIA was then modified to replace the bacterialgus (β-glucoronidase) marker gene with the Venus™ fluorescent markergene (Clontech, Mountain View, Calif., USA) to improve the speed of rMVAscreening. The CMV pp65 gene was cloned into mH5-pZWIIA to yieldmH5-pp65-pZWIIA. The IEfusion gene was cloned into mH5-pZWIIA to yieldmH5-IEfusion-pZWIIA, and an MVA transfer plasmid was used to generatemH5-IEfusion-MVA. To make rMVA expressing both pp65 and IEfusion proteinsimultaneously, a new MVA transfer vector that contained mH5 promoterand targets MVA deletion III region was constructed based on the pLW51plasmid. pLW51 was then modified by replacing the original expressioncassette by excision at XhoI and AscI sites and inserted the mH5promoter followed by the CMV pp65 gene to yield mH5-pp65-pLW51. Thestructure of MVA transfer vectors (mH5-pp65-pZWIIA, mH5-IEfusion-pZWIIAand mH5-pp65-pLW51) were verified by restriction enzyme digestion andDNA sequence analysis.

mH5-pp65-MVA was generated by transfecting mH5-pp65-pZWIIA intowtMVA-infected BHK-21 cells and screened based on the Venus™ fluorescentmarker to eliminate wtMVA as previously described (Wang et al. 2006).mH5-pp65-IEfusion-MVA was generated in two steps. First,mH5-IEfusion-MVA was generated by transfecting mH5-IEfusion-pZWIIA intoBHK-21 cells infected with wtMVA in six-well plates. mH5-IEfusion-MVAwas screened to eliminate wtMVA based on Venus™ fluorescent markerexpression. mH5-IEfusion-MVA was expanded on BHK-21 cells after 8-10rounds of screening to create a stock for the 2nd round of geneinsertion after verification that wtMVA was eliminated. Next,mH5-pp65-pLW51 was transfected into BHK-21 cells that weresimultaneously infected with mH5-IEfusion-MVA. mH5-pp65-IEfusion-MVA wasscreened based on the bacterial gus gene marker for 8-10 rounds untilparental virus (mH5-Iefusion-MVA) was removed completely. mH5-pp65-MVAand mH5-pp65-Iefusion-MVA were expanded on BHK-21 cells to create virusstocks that were stored long term at −80° C.

Stability Analysis of Individual rMVA Isolates from Passage 10.

rMVA with expression cassettes based on mH5 (mH5-pp65-MVA,mH5-pp65-IEfusion-MVA) or pSyn promoters (pSynpp65-IE1/e4-MVA,pSyn-pp65-IEfusion-MVA) were consecutively passaged 10 times on eitherCEF or BHK-21 cells. Briefly, a 150 mm tissue culture dish of either CEFor BHK-21 cells was infected with rMVA at multiplicity of infection of0.1 (MOI=0.1). rMVA was harvested 48 h after infection, resuspended in1.0 ml of MEM containing 2% fetal calf serum (MEM-2) and subjected to 3×freeze/thaw cycles followed by sonication to release the virus. Thevirus from each passage was subsequently titrated on either CEF orBHK-21 cells and after adjustment to an MOI of 0.10, it was used for thenext passage. DNA samples of each passage were obtained for qPCRanalysis using the Qiagen™ column purification kit according tomanufacturer's instructions (Valencia, Calif., USA). Cell lysates ofeach passage used for Western blot (WB) analysis were prepared from 100mm dishes of either CEF or BHK-21 cells infected with the same number ofpfu of rMVA of each serial passage.

To further characterize virus plaques from passage 10 (P10) ofpSyn-pp65-IE1/e4-MVA, individual plaques were isolated from P10 virusstock by plaque purification. Briefly, P10 virus stock ofpp65-IE1/e4-MVA (pSyn) was titrated by immunostaining usinganti-vaccinia polyclonal sera (AbD serotech, Raleigh, N.C., USA),diluted and distributed at 0.5 pfu per well into 96-well plates. At 4days post-infection, 18 wells that appeared to be infected by no morethan one virus isolate were collected, expanded and analyzed by WB forCMV-pp65 expression levels. Six individual plaques from P1 were alsoisolated at random using the same method.

Western Blot (WB) Detection of rMVA Protein Expression.

Protein expression levels of the pp65, IE1/e4 and IEfusion genes frompSyn-pp65-IE1/e4-MVA, pSyn-pp65-IEfusion-MVA and mH5-pp65-IEfusion-MVAinfected cells were measured by Western blot using the Amersham ECLPlus™ detection kit (Amersham Pharmacia Biotech, Buckinghamshire, UnitedKingdom). Cell lysates were separated by SDS-PAGE on 10% gels. Afterelectro-transfer of proteins from the gel onto PVDF membranes (Bio-Rad,Hercules, Calif.), the blots were incubated with purified mAb 28-103(against pp65) or mAb p63-27 (against IE1), then washed and furtherincubated with HRP-labeled goat anti-mouse Ab according to themanufacturer's instructions (Amersham Pharmacia Biotech™).

Southern Blot Detection of CMV-pp65 and IE1/e4 Insertion Gene in rMVAs.

To determine the presence of the pp65 and IE1/e4 gene in individualpp65-IE1/e4-MVA isolates after P10, southern blot (SB) was performed.Briefly, a 150 mm culture dish of BHK-21 cells was infected withindividual pp65-IE1/e4-MVA isolates at MOI=1 and incubated at 37° C. for24 hours. The MVA viral genomic DNA was isolated according to adescribed method (Cwynarski et al. 2001). Briefly, cells werehomogenized in 1.25 ml hypotonic buffer (10 mM Tris-HCl, pH 7.8containing 12 mM KCl followed by incubation with 450 units ofmicrococcal nuclease (Sigma-Aldrich St. Louis, Mo.) for one hour at 25°C. to digest cellular DNA. The reaction was stopped by adding EGTA(glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Cell lysateswere treated with proteinase K for one hour at 25° C. to release MVAviral DNA and then extracted using the phenol/chloroform method. For SB,MVA viral DNA was digested with Pme I and Nhe I restriction enzymes toexcise the 3.9 Kb fragment containing the foreign gene cassette,separated on a 1% agarose gel and transferred to nylon membrane. Thisfilter was hybridized with a ³²P-labeled DNA probe specific for bothpp65 and IE1 exon4 gene and exposed to HyPerfilm (Amersham Bioscience,Piscataway, N.J. 08855).

qPCR to Measure DNA Copy Number.

MVA viral DNA was extracted using a Qiagen QIAmp miniprep kit accordingto the manufacturer's instructions (Qiagen, Valencia, Calif.). Theplasmid DNA used to generate the standard curve was made by insertingboth the pp65 and IEfusion gene into the pSC11 vector containing the TKgene (La Rosa et al. 2002). The absolute concentration of the plasmidwas measured by two independent means: OD₂₆₀ by UV spectrophotometry anda fluorophore-based method using Quant-iT™ Picogreen® dsDNA kit(Invitrogen™, Carlsbad, Calif., USA). The concentration was converted toplasmid copy number using the molecular weight of the plasmid DNA.Quantitative PCR primers of target genes were designed based on standardqPCR conditions using Primer Express Software Version 3.0 (AppliedBiosystems Inc., Foster City, Calif., USA) and listed in Table 1, below.Quantitative PCR was performed using an ABI 7300 real-time PCR systemand Power SYBR green master mix (SYBR) kit (Applied Biosystems).Briefly, 5 mL of MVA genomic DNA was amplified in a mixture of 25 μLcontaining 1 μM forward, 1 μM reverse primers and SYBR solution. Thethermal cycling conditions were 95° C. for 10 min, 40 cycles of 95° C.for 15 seconds, and ending with one cycle at 60° C. for 30 seconds. Genecopy numbers were calculated using ABI sequence detection systemsoftware (SDS). The ratio of insert CMV genes and MVA backbone(ratio=CMV gene copy number/MVA backbone computed copy number) wascalculated for each passage.

TABLE 1 Quantitative PCR Primers. Forward or SEQ ID Name ReverseSequence NO pp65 Forward 5′ ATCAAACCGGGCAAGATCTCGC 3′ 1 pp65 Reverse 5′ATCGTACTGACGCAGTTCCACG 3′ 2 IE1 exon4 Forward 5′ CCATCGCCGAGGAGTCAGAT 3′3 IE1 exon4 Reverse 5′ AGTGTCCTCCCGCTCCTCCT 3′ 4 IEfusion Forward 5′AAGTTGCCCCAGAGGAAGAG 3′ 5 IEfusion Reverse 5′ CTGCTAACGCTGCAAGAGTG 3′ 6TK Forward 5′ TGTGAGCGTATGGCAAACGAA 3′ 7 TK Reverse 5′TCGATGTAACACTTTCTACACACCGATT 3′ 8

Immunogenicity of mH5-pp65-IEfusion-MVA Using Human PBMC.

CMV-positive healthy volunteers were enrolled in an IRB-approvedprotocol with informed consent. Whole blood was collected Humanperipheral blood mononuclear cells (PBMC) were collected, purified usingFicoll™ and cryopreserved at −80° C. All human blood samples wereconsidered discard and anonymous, except for HLA A and B typinginformation provided to investigators without other identifiers. IVS ofPBMC using rMVA was performed according to previously described methods(La Rosa et al. 2006, Wang et al. 2007). Briefly, cryopreserved PBMCwere rapidly thawed and cultured with both CpG-A ODN 2216 and CpG-B ODN2006 (TriLink BioTechnologies, San Diego, Calif., USA). After 3 days,PBMCs were infected with rMVA for 6 hours, γ-irradiated (2500 rads) andused as APC incubated with autologus PBMC for 7 days.

PBMC that were harvested at 8 days post-IVS and incubated with eitherCMV-pp65 or IEfusion peptide library for 12 hours in the presence ofbrefeldin A, then washed and labeled with PE-conjugated anti-CD8 andFITC-conjugated anti-CD4 antibodies, fixed, and permeabilized(Cytofix-Cytoperm; BD Biosciences) before they were labeled withAPC-conjugated antibody to IFN-γ. The stained cells were analyzed on aFACSCanto™ flow cytometer (BD Biosciences). Comparison of paired databetween P1 and P7 of mH5-pp65-IEfusion-MVA was performed using thestudent t-test based on two-tailed procedure. The P values wereconsidered significant if <0.05.

Immunogenicity of rMVA in HHD II Tg Mice.

Immunogenicity of mH5-pp65-mH5-IEfusion-MVA of passage 1 and 7 also wastested in HHD II mice (HLA A2.1). HHD II mice (Tg HLA A2.1) were used at6-12 weeks for immunization and were bred and maintained under SPFconditions in a centralized animal care facility. HHD II mice wereimmunized with 5×10⁷ pfu of purified rMVA intraperitoneally (i.p.).Spleens were removed three weeks after immunization and were stimulatedin vitro for one week using a simplified protocol with HLA-matched humanEBV-LCL (Krishnan et al. 2008) as antigen presenting cells (APC), loadedeither with the relevant CMV-CTL epitope HLA-A*0201 IE-1₃₁₆₋₃₂₄(IE1-A2), pp65₄₉₅₋₅₀₃ (pp65-A2) (Wills et al. 1996; Diamond et al. 1997;Khan et al. 2002) or IE2 CMV-peptide library (4 μg/ml) as describedabove.

ICC was used to measure pp65, IE1 and IE2 IFN-γ⁺/CD4⁺ or IFN-γ⁺/CD8⁺ Tcells from the stimulated splenocytes according to methods previouslydescribed (La Rosa et al. 2001; La Rosa et al. 2006; Cobbold et al.2005; Cosma et al. 2003). 0.5 to 1×10⁶ events were acquired for eachsample on a FACSCanto™ (BD Biosciences, San Jose, Calif.). Analysis wasperformed using FCS Express version 2 software (De Novo, Ontario,Canada). The number of double-positive cells was expressed as apercentage of the CD8⁺ T-cell population.

Comparison of mH5 and pSyn Promoter Activity in rMVA Infected Cells.

To determine early or total transcriptional activity of mH5 and pSynpromoters in rMVA infected cells, pp65 expression levels were determinedin BHK-21 cells that were infected with mH5-pp65-MVA or pSyn-pp65-MVA inthe absence or presence of cytosine arabinoside (Ara-C) by quantitativeWestern blot. BHK-21 cells were seeded at 0.6×10⁶ per well onto a 6-wellplate. The cells were infected with either mH5-pp65-MVA or pSyn-pp65-MVAat MOI=5 in the presence or absence of 40 μg/ml of Ara-C and incubatedfor 24 hours at 37° C. in a 5% CO2 incubator. The infected cells wereharvested at 24 h post-infection and lysed in SDS-PAGE loading buffer(62.5 mM Tris-HCl, pH 6.8, 2.8 mM β-mercaptoethanol, 2% SDS, 10%glycerol, 0.4% Bromophenol Blue). Cell lysates were separated bySDS-PAGE on 10% gels. After electro-transfer of proteins from the gelonto PVDF membranes (Bio-Rad, Hercules, Calif., USA), the blots wereincubated with purified mAb 28-103 (Britt et al. 1985) against pp65 andmAb against β-tubulin (Sigma-Aldrich), and then washed and furtherincubated with HRP-labeled goat anti-mouse Ab according to themanufacturer's instructions. pp65 protein expression was measured byincubating the blots with chemifluorescence substrate solution in ECLPlus detection kit (Amersham, Calif.) for 30 minutes and were scannedusing Typhoon™ 9410 workstation and analyzed using ImageQuant TLsoftware (GE Healthcare Bio-Sciences Corp, Piscataway, N.J., USA).β-tubulin was used as internal control for each lane.

Pulse-Chase Metabolic Labeling and Immunoprecipitation.

Pulse-chase (PC) and immunoprecipitation (IP) were performed based onmodification of described methods (Tobery et al. 1997; Wang et al.2004b). Briefly, subconfluent cultures of CEF or BHK-21 cells grown in6-well plates were infected at an MOI of 10 with mH5-pp65-MVA orpSyn-pp65-MVA. At 1 hour postinfection (hpi), cells were washed andincubated with Cys-free and Metfree DMEM (Invitrogen, Carlsbad, Calif.,USA) medium containing 5% dialyzed fetal calf serum (FCS; Invitrogen)for 1 hour. Cells were then metabolically labeled (100 μCi/mL/well) for30 min with a mixture of [³⁵S]Cys+[³⁵S]Met [Express Protein LabelingMix™ (1000 Ci/mmol) PerkinElmer, Boston, Mass., USA]. After labeling,the cells were washed twice with PBS and either harvested immediately orchased in RPMI medium with 10% FCS (ISC-BioExpress, Kaysville, Utah,USA) supplemented with excess unlabeled methionine (1 mM) and cysteine(5 mM) up to 10 hours. After each time point, cells were immediatelypelleted, then lysed in 1.0 mL PBS containing 1.0% Triton X-100, 1.0%sodium deoxycholate (Sigma, St. Louis, Mo., USA) and 0.1% SDS in thepresence of Protease Inhibitor Cocktail (Roche, Nutley, N.J., USA).Supernatants (0.5 mL) were precleared once with 50 μL of proteinA/G-agarose beads (Santa Cruz Biotechnology) for 1 h. Sequentialincubation with 2.4 μg purified mAb against CMV-pp65 (mAb 28-103; Brittet al. 1987) was followed by an isotope-specific mAb (19C2; Schmeiz etal. 1994) for 2 hours. Immune complexes were captured by incubation for1 hour with 50 μL of protein A/G beads. The immune complex bound ProteinA/G beads were washed 4 times with 0.1% Triton X-100 in PBS and boundproteins were eluted by boiling in 0.2% SDS, 5 mM DTT, 40 mM sodiumphosphate buffer (pH 6.5) into SDS-polyacryamide gel electrophoresis(PAGE) sample buffer. Proteins were separated by 10% SDS-PAGE anddetected by autoradiography using X-OMAT film (Kodak, Rochester, N.Y.,USA).

Serial Passage of pSyn-pp65-IE1/e4-MVA

pp65-IE1/e4-MVA (pSyn) was generated using a pZWIIA transfer plasmid aspreviously described (Wang et al. 2007). The pp65 and IE1/e4 geneexpression cassettes were integrated into the deletion integration siteII of the MVA genome (Del II) via homologous recombination as shown inFIG. 1A. pSyn-pp65-IE1/e4-MVA was sequentially passaged for 10 rounds onprimary chicken embryo fibroblast (CEF) cells prepared from specificpathogen-free chicken eggs. The virus titer and growth rate of eachpassage was measured. There was no significant change in virus titer andgrowth rate during serial passage. Cell lysates of each passage wereprepared in parallel from 100 mm culture dishes of cells infected withthe same amount of virus established by titration on CEF cells.

FIG. 1B shows Western blot detection of pp65 and IE1 exon4 expressionlevels of pp65-IE1/e4-MVA-infected CEF cells (serial passages p1 top10). pp65 and IE1/exon4 protein levels progressively decreased duringpassage, and were significantly reduced after ten serial passages. Thetop panel was blotted with mAb 28-103 specific for pp65 to determinepp65 expression levels; the middle panel was blotted with p63-27specific for IE1 exon4 to determine IE1 exon4 protein expression levels.The bottom panel of FIG. 1B shows the constitutively expressed MVAprotein BR5, which was also proved at each passage from the same lysatesusing the 19C2 mAb (Schmeiz et al. 1994), The steady state expressionlevel of BR5 was unchanged during the 10 passage evaluation. Serialpassage of pSyn-IE1/e4-MVA was also carried out on CEF cells withsimilar results.

Preparation and Expression Analysis of 18 Individual P10 Isolates

To determine whether gradual decrease of the pp65 and IE1 expressionlevels during serial passage can be caused by genetic changes thatresult in non-expressing variants, individual isolates were isolatedfrom passage 10 (P10) by plaque purification. Eighteen wells thatappeared to have cyto-pathologic effects (CPE) were collected at 4 dayspost-infection. Each virus sample was considered to be a single isolatebecause the equivalent of 0.5 pfu of virus was distributed in each well.Viral infection from these collected samples was confirmed by continuousvirus growth and virus titration. Thus, eighteen individualpSyn-pp65-IE1/e4-MVA viruses were isolated from passage 10 by virusplaque purification and expanded in CEF cells to prepare cell lysatesfor Western blot. See FIG. 1C. As illustrated in FIG. 1C, eight of the18 (40%) collected individual isolates had lost pp65 expression. Incontrast, six of six (100%) individual isolates from P1 all had similarstrong levels pp65 expression. Each lane of FIG. 1C contains a singleindividual isolate from passage 10. Samples #4, #6, #7 and #13 (markedwith a star in the figure) were selected for further analysis asdescribed below.

Deletion of the pp65 and IE1/e4 Gene was the Cause of Loss of pp65 andIE1/e4 Protein Expression from Individual Virus Isolates ofpSyn-pp65-IE1/e4-MVA.

To determine whether mutations or total deletion of the pp65 and IE1genes during serial passage were responsible for this loss of proteinexpression, two of the isolates described above with full expressionlevels, two isolates that lost pp65 protein expression (#4, #6 in FIG.1C) and two isolates that retained pp65 expression from P10 (#7, #13 inFIG. 1C) were selected. Western blot was performed on these isolates todetect both pp65 and IE1 protein expression levels, and Southern blotwas used to detect pp65 and IE1 expression cassettes from viral DNA.

A monoclonal antibody specific for an MVA viral protein (BR5) wasincluded in the Western blot to detect endogenous viral gene expressionto control for virus input in all six samples. See FIG. 2A, panel (iii).The two individual isolates from passage 10 that maintained pp65expression also expressed IE1 at similar level as P1. (FIG. 2A, panel(i)) In contrast, the two isolates from P10 that lost pp65 expressionalso coordinately lost IE1 protein expression (FIG. 2A, panel (ii)). Allfour cases showed either coordinate expression of both antigens or theirabsence, suggesting that the whole cassette was either maintained orinactivated by deletion or mutation when protein expression was notdetected. In contrast, the expression of the MVA BR5 protein remaineduniformly unchanged (FIG. 2A, panel (iii)).

A Southern blot detected the pp65 and IE1 genes and established therelationship of protein expression levels and the presence of the genes.Equal amounts of DNA from each viral isolate was digested with Pme I andNot I restriction enzymes to excise pp65 and IE1/e4 gene expressioncassettes (3.9 Kb), which were detected by a ³²P-radiolabeled DNA probe.The gene expression cassette was detected in two individual virusisolates from P1 and P10 (lanes 1, 2, 5, 6 in FIG. 2B), but not detectedin two viral isolates from P10 that also lost protein expression (Lane 3and 4 in FIG. 2B). The del II site of MVA was further analyzed by DNArestriction endonuclease analysis of MVA genomic DNA and by PCR using aseries of primers that target the surrounding del II region. CMV-pp65and IE1 gene expression cassettes together with the surrounding MVA delII region were found to be absent. The possibility that the twonon-expressing mutants were contaminant wild type MVA virus that wasintroduced and amplified during the serial passage was excluded usingadditional qPCR primers. Expression of pp65 and IE protein wascorrelated with the presence of the corresponding genes, suggesting thatlarge deletions rather than small ones resulted in their absence.

Two isolates from P10 maintained pp65 and IE expression levels as P1(FIG. 2A, lanes 1 and 2). These isolates were tested to determinewhether they represented stable forms of pSyn-pp65-IE1/e4-MVA and couldmaintain stable expression of both insert genes during serial passage.These two isolates were sequentially passaged for an additional 10rounds on CEF cells. Both pp65 and IE1 protein expression stilldecreased to a low level at the conclusion of additional serial passage.These results demonstrate that high-expressing isolates from P10 are notstabilized forms of pSyn-pp65-IE1 exon4-MVA, and are subject to deletionduring passage.

Immunogenicity of pSyn-pp65-IE1/e4-MVA is Reduced after Serial Passage.

To determine if reduction of pp65 and IE1 protein expression impactedimmunogenicity, P1 and P10 virus stocks were expanded for mouseimmunizations. HHD II mice (Tg HLA A2.1) were used at 6 to 12 weeks forimmunization and were bred and maintained under SPF conditions in acentralized animal care facility. Human peripheral blood mononuclearcells (PBMC) were collected, purified using Ficoll™ and cryopreserved at−80° C. HHD II mice were separately immunized with both P1 and P10passage strains for 3 weeks.

Splenocyte immune response was assessed by ICC to detect IFN-γexpression. Immunodominant HLA A2-restricted pp65 and IE1 CTL epitopeswere used to evaluate the HLA A2-restricted CD8+ T cell response. SeeFIG. 3. The results show a statistically significant diminution of pp65and IE1 specific-INF-γ producing CD8 positive T cells between P1 and P10immunized groups.

Genetic Stability of pSyn-pp65-IE1/e4-MVA Measured by qPCR

Since progressive loss of pp65 and IE1 protein expression is correlatedwith the deletion of gene expression cassettes, the kinetics of the lossof the genes was measured to develop a potential mechanism. The geneticstability of rMVA can be assessed by computing the ratio of the geneinsert and the MVA backbone copy number.

The ratio of gene insert to MVA backbone at initial passage wasnormalized to unity, and a gradual reduction during serial passage. Only20% of the rMVA retained pp65 and IE1 exon4 gene inserts after roundP10. See FIG. 4A. This measurement establishes a correlation between thedisappearance of foreign protein antigen genes that is confirmed byqPCR, lower protein expression levels and reduced immunogenicity of thepassaged viral population.

Genetic Stability of pSyn-pp65-IEfusion-MVA Measured by qPCR

Recombinant MVA expressing three CMV antigens under control of pSynpromoters (pSyn-pp65-IEfusion-MVA) were constructed to expand therepresentation of early genes and epitope according to methods asdescribed in Example 1. pSyn-pp65-IEfusion-MVA includes the IE2-exon5gene which is fused to IE1-exon4. pSyn-pp65-IEfusion-MVA viral genomicDNA was extracted and qPCR was performed using pp65, IEfuson and TKspecific primers as described herein.

pSyn-pp65-IEfusion-MVA was serially passaged five times. Even after asingle passage, however, evidence of instability was observed (FIG. 4B).Only 10% of the original levels of pp65 and IEfusion insert sequenceswere detected by qPCR after 5 passages, which demonstrates an unexpecteddecrease in stability, possibly because of the gene fusion. See FIG. 4B.This result highlights that different combination of genes (pp65 andIE1/e4 and pp65 and IEfusion) result in pronounced genetic instabilityusing the pSyn promoter, suggesting that the genes themselves are notthe main contributor to genetic instability compared to the activity ofthe pSyn promoter.

Construction of mH5-pp65-MVA and Measurement of Genetic Stability

Although the pSyn promoter was optimized for high level proteinexpression and was designed to be highly active by combining severalearly and late promoter elements, it is dominated by its late stagepromoter activity (Chakrtabarti et al. 1997). Therefore the instabilityof pSyn-pp65-IE1/e4-MVA and pSyn-pp65-IEfusion-MVA may be due to theproperties of pSyn promoters. To improve genetic stability, the pSynpromoter was replaced with the mH5 promoter which stimulates a greaterproportion of its transcriptional activity at an earlier stage of thevirus life cycle (FIG. 5A) (Wyatt et al. 1996; Earl et al. 2009). rMVAwas generated using shuttle plasmids that had the mH5 promoter directingthe transcription of the CMV-pp65 gene. Quantification by qPCR revealedno significant changes in the ratio of CMV insert gene/MVA backbonegenomic copy number during 10 serial passages of a virus using the mH5promoter directing recombinant protein expression (FIG. 5B).

Genetic Stability of rMVA Expressing CMV-pp65 and IEfusion under mH5Promoter Control

A single rMVA simultaneously expressing both CMV-pp65 and IEfusionproteins was constructed using dual mH5 promoters using two strategies.First, an MVA expressing all three foreign protein antigens wasconstructed by targeting a single integration site (del II) with aplasmid shuttle vector that had tandem mH5 promoters in opposingorientation. It could not be stably prepared, likely due tointramolecular homologous recombination, that is presumably initiated bythe identical mH5 promoter copies. Second, the CMV-pp65 and IEfusiongenes were inserted at two separate sites in MVA (del II located at149,261 and del III located at 20,625 of the MVA genome) to prevent thedeletional recombination mediated by the two identical copies of the mH5promoter. A schematic picture of the structure of this rMVA and theinsertion sites is provided in FIG. 5A. This virus was successfullyconstructed, and passaged 10 times in a similar manner as was done forthe pSyn viruses above (FIGS. 4A and 4B). The passages were conducted onboth BHK-21 (FIG. 5C) and CEF (FIG. 5D) cells. Genetic stability wasevaluated by qPCR using three primer pairs specific for the CMV-pp65 andIEfusion genes, and the MVA viral genomic backbone, respectively. TheqPCR results for both CMV antigens are computed as a ratio to the viralgenomic MVA backbone (FIG. 5C). Both CMV gene inserts at del II and IIIintegration sites had excellent stability, with almost 100% of each genecopy number maintained after 10 passages compared to P0 (FIG. 5C). Asimilar result was found with virus passaged on CEF, using the CMV-pp65and the MVA backbone sequences as targets for qPCR (FIG. 5D).

Target sequences measured by qPCR represent a small region (0.2-0.3 bps)of CMV-pp65 (1.7 kb) and IEfusion gene (2.9 kb) insertion. To excludethe possibility that the qPCR results may not represent focused regionsof instability throughout the entire length of both genes, severaladditional pairs of primers targeting different regions of CMV-pp65 andIEfusion gene were designed. The ratio of CMV-pp65 or IE1 or IEfusioncompared to the MVA genomic DNA backbone was similar throughout thelength of each insert gene.

Minimal Change in Immunogenicity of mH5-pp65-IEfusion-MVA after SerialPassage

To determine if genetic stability of mH5-pp65-IEfusion-MVA after 10multiple passages translated to equivalent immunogenicity at passage P1and P7, the capacity of both the P1 and P7 passage viral stocks tosupport vigorous amplification of a memory T cell response afterexposure of human PBMC to MVA vaccines was assessed (Wang et al. 2004b).Both P7 and P1 passages (p=NS by Student t-test) (FIG. 6A) showedequivalent immunogenicity. The qualitative differences between T cellsubsets stimulated by individual foreign protein antigens were notaltered after 7 passages in peripheral blood mononuclear cells (PBMC)from four healthy volunteers. There also was no significant difference(p>0.5, paired t test) in the response of HHD II mice immunized with themH5-pp65-IEfusion MVA virus stocks at passages P1 and P7 similar indesign to experiments described above and shown in FIG. 3. Very highlevels of CMV pp65-specific, IE1-specific and IE2-specific IFN-γ ⁺CD8⁺ Tcells were found, confirming the equivalence of P1 and P7 viral passagesstates at eliciting high-level immunogenicity in all immunized mice. SeeFIG. 6B.

Early Expression of CMV-pp65 is Stronger Under Control of mH5 Promoterthan pSyn Promoter While Late Expression Levels are Similar

Ara-C (cytosine β-D-arabinofuranoside) is a deoxycytidine analog whichincorporates into DNA and inhibits DNA replication by forming cleavagecomplexes with topoisomerase I resulting in DNA fragmentation [Azuma etal. 2001]. It is a selective inhibitor of DNA synthesis that does notaffect RNA synthesis in mammalian cells [Dawson et al. 1986] and so canbe used to distinguish early and late protein expression in cells andthe timing of transcriptional activation of the mH5 and pSyn promoters.Cell lysates prepared from rMVA infected cells in the absence of Ara-Ccontained both early and late pp65 protein expression, however, celllysates prepared from rMVA infected cells in the presence of Ara-Ccontain only early expression of pp65 protein because DNA replicationand late gene expression were blocked by Ara-C.

Quantitative WB employing β-tubulin was used as an internal standard tocompare CMV-pp65 expression levels in lysates from cells infected witheither mH5-pp65-MVA or with pSyn-pp65-MVA in the absence or presence ofAra-C. In the absence of Ara-C, similar CMV-pp65 protein expressionlevels were observed in both mH5-pp65-MVA-infected and inpSyn-pp65-MVA-infected cells. However, in the presence of Ara-C, therewas a 7-fold higher level of CMV-pp65 expression in cells infected withmH5-pp65-MVA as compared to cells infected with pSyn-pp65-MVA (Table 2).As shown in Table 2 below, early pp65 expression in mH5-pp65-MVA(+Ara-C) accounted for 40% of total pp65 expression (−Ara-C) while earlypp65 expression in pSyn-pp65-MVA (−Ara-C) accounted only for 6% of totalpp65 expression (−Ara-C).

TABLE 2 Early and late activities of mH5 and pSyn promoters as measuredby quantitative Western blot. Insert pp65 expression pp65 expressionratio Promoter gene +Ara-C −Ara-C (−Ara-C/+Ara-C) mH5 pp65 0.9 2.25(2.25/0.90) pSyn pp65 0.13 2.23 (2.23/0.13)

Pulse-Chase Analysis Reveals Equal Protein Stability of CMV-pp65 AntigenUnder the Control of Either pSyn or mH5 Promoters.

Alternative explanations for the difference in stability of MVA virusesthat utilize the pSyn or mH5 promoter originally demonstrated by areduction of specific signal from the CMV-pp65 and IE1/e4 protein bands(FIGS. 1 and 2) were explored. To determine whether the reduction inexpression can be explained by differential protein stability when thepSyn promoter is used, rather than timing of expression, a pulse-chaseapproach was used. In this approach, MVA-infected CEF (FIG. 16) andBHK-21 cells (data not shown) were metabolically radio-labeled, followedby cold chase to measure the disappearance of radio-labeled CMV-pp65protein, which is a measure of its stability to degradation.

The pulse-chase approach used in these studies was similar to previousapproaches (Wang et al. 2004b). Three time points of cold chase through10 hours were utilized, as this time frame is sufficient to measuredifferences in protein stability based on prior work with CMV-pp65. Theinfection conditions of CEF and BHK-21 cells were similar as those usedfor the analysis of protein expression in FIGS. 1 and 2. The change inlabeled CMV-pp65 is limited over the first 4 hours of chase, with only aminimal decline at the 10 hour time point for both promoter constructs(FIG. 7). The pattern of CMV-pp65 expression and stability is equivalentwhen either the mH5 or pSyn constructs were evaluated. The specificityof the recognition of radiolabeled CMV-pp65 is shown by the absence ofan equivalent CMV-pp65-specific radiolabeled band in the gus-MVAinfected control lane. Similar to previous studies, two closelyjuxtaposed bands are found after immunoprecipitation (IP) with mAb28-103. Based on the differences between the CMV-pp65 decay profile andthe non-specific band, the lower band is likely to reflect the targetpp65 protein. Moreover, examining the same extracts using an isotypecontrol mAb shows absolute specificity for the pp65 protein (data notshown). Therefore, the choice of promoter does not dramaticallyinfluence the degradation rate of the CMV-pp65 antigen. Consequently,protein stability is likely not a factor in determining the stabilitycharacteristics of both MVA expressing CMV-pp65.

EXAMPLE 3 Generation and Expansion of pp65-IEfusion-MVA (CMV-MVA) VirusSeed for Large Scale GMP Production

The genetic stability of the recombinant virus is a concern for viralvector based vaccines intended for clinical investigation, because theymust be amplified multiple times to reach the scale needed for cGMPmanufacturing process (Wyatt et al. 2009; Earl et al. 2009). The vectormust retain its potency to fulfill expectations of regulatory agenciesincluding FDA that require the manufacturing process not irrevocablyalter the virus structure or the potency of the vaccine. Geneticallystable pp65-IEfusion-MVA virus seed was generated and tested accordingto the examples above and was further characterized to optimize virusproductivity and to establish feasibility for its use in large scale GMPproduction.

mH5-IEfusion-pZWIIA (GUS) shuttle plasmid (as described above) wasgenerated using an endotoxin-free preparation (Qiagen) and was verifiedby restriction enzyme digestion (AscI and Pme I) and DNA sequenceanalysis (FIG. 19). The mH5-IEfusion-pZWIIA (GUS) was transfected intoMVA 572.FHE-22.02-1974 infected primary CEF cells and screened based onthe gus marker gene. Ten independent isolates (R10 isolates) wereselected for the first round of plaque isolation and were screened forIEfusion antigen by immunostaining using anti-CMV IE1 mAb (p63-27). Thefive isolates having the highest expression in the first round wereselected for the second round of plaque isolation. Ten rounds of plaqueisolation were conducted, using five isolates at each successive round.At rounds 3, 6, 8 and 10, qPCR using primers shown in Table 1 above wasperformed to determine gene copy numbers of IEfusion and contaminatingwtMVA. Gene copy numbers were determined using SYBR Green as a reporter.An IEfusion standard curve was established using plasmid copy numbersfrom 10² to 10⁷ (FIG. 29). Each isolate was then measured against thestandard curve. An exemplar amplification plot for R10 isolate sample8B1A1A1A (9.00E+07 copies; FIG. 29) is shown in FIG. 20. The qPCRResults for IEfusion-MVA R10 isolates are shown in Table 3 below.

TABLE 3 qPCR Data for IEfusion-MVA R10 Isolates R10 Isolates IEfusioncopy # wt copy # MVA backbone # 7A2B2B1B1C 1.59E+07 undetected 1.74E+077A2B2B1B1C 1.64E+07 undetected 1.94E+07 7A2B2B1B1D 5.93E+07 undetected1.03E+08 7A2B2B1B1D 6.21E+07 undetected 9.54E+07 8B1A1B1B1A 7.02E+06undetected 1.99E+07 8B1A1B1B1A 6.54E+06 undetected 1.66E+07 8B1A1A1A(R8) 9.00E+07 undetected 2.22E+08

Four wt-free MVA isolates (8B1A1A1A, 8B1A1B1B1A, 7A2B2B1B1C and7A2B2B1B1D) were expanded to create candidate expanded stocks. Thestocks were then further characterized for microbial contamination, andwere analyzed by Western blot for detection of IEfusion antigen andvirus titer (FIG. 21). The gus marker gene was then removed by limitingdilution from two of the candidates (8B1A1B1B1A and 7A2B2B1B1D), andnon-blue samples were screened by immunostaining to verify IEfusionexpression. Samples that were positive for IEfusion were analyzed byqPCR for the presence of the IEfusion gene and absence of gus and wt-MVAgenes. One sample that was confirmed to be gus marker gene-free,wt-MVA-free and had a high IEfusion gene copy number was selected andthe selected expanded stock and further characterized for microbialcontamination, IEfusion protein expression by Western blot (FIG. 22),and titer. The IEfusion-MVA was sequenced (SAIC-F COTR) to verify thatno point mutations occurred.

After the IEfusion-MVA virus seed was established, pp65-IE-fusion-MVA(CMV-MVA) was generated. Briefly, CEF cells were simultaneously infectedwith IEfusion-MVA generated in the first step and mH5-pp65-pLW51 shuttleplasmid that was verified by restriction enzyme digestion (Asc I and PmeI) and DNA sequence analysis (FIG. 25). The co-infected CEF cells werescreened based on the gus marker gene. Eight rounds of plaque isolationwere performed, and 15-20 plaques (gus+) were selected at each round.

The plaques were immunostained at each round using mAb against IE(p63-27) and pp65 (28-103). After the eighth round (R8), eighteensamples were characterized by qPCR for absence of parental MVA(IEfusion-MVA) and for detection of IEfusion, pp65, MVA backbone copynumbers, and candidates for expansion are shown in Table 4 below. Alleighteen samples were determined to be parental MVA-free, and detectionof IEfusion (FIG. 26A) and pp65 (FIG. 26B) was confirmed.

TABLE 4 pp65-IEfusion-MVA: Candidates for Expansion MVA R8 IEfusion pp65backbone Deletion III IE:pp65 IE:backbone pp65:backbone Sample ID copy #copy # copy # copy # copy ratio copy ratio copy ratio 14B1C2A 1.48 × 10⁷9.65 × 10⁶ 9.15 × 10⁶ Undetected 1.5 1.6 1.05 3B 14B1C2E 7.81 × 10⁶ 7.94× 10⁶ 5.01 × 10⁶ Undetected 0.98 1.56 1.58 4B 14B1C2E 1.23 × 10⁷ 1.10 ×10⁷ 1.19 × 10⁷ Undetected 1.1 1.03 0.92 7C 14B1C2F 1.27 × 10⁷ 1.65 × 10⁷1.28 × 10⁷ Undetected 0.77 0.99 1.29 1B

The gus marker was removed from two candidates by limiting dilution, andnon-blue samples for IE and pp65 were immunostained for antigenexpression. Samples that were positive by immunostaining for both IE andpp65 were characterized by qPCR for absence of gus and presence ofIEfusion and pp65. Two samples that that had equivalent copy numbers ofIEfusion and pp65 and were gus marker gene-free and parental MVA-freewere identified (F8 and 23D5) and the two pp65-IEfusion-MVA wereexpanded. The expanded pp65-IEfusion-MVA were completely characterizedfor microbial contamination, and were analyzed by Western blot fordetection of IEfusion (FIG. 27A) antigen, pp65 (FIG. 27B) antigen andvirus titer.

For large-scale expansion, twenty-five T-175 flasks were used togenerate the CMV-MVA seed for the expanded sample F8, which expressedboth IEfusion and pp65 described above. Complete characterization wasaccomplished by plaque assay titration, detection of IEfusion (FIG. 28A)and pp65 (FIG. 28B) by Western blot, host cell restriction, microbialand mycoplasma contamination tests, and sequence identity. The CNV-MVAvirus seed was negative for microbial and mycoplasma contaminationtests, the CMV-MVA virus seed titer was 1.95×108 pfu/ml, and thesequence identity of the virus seed was confirmed by SAIC-F COTR. Use ofthe CMV-MVA seed described herein for large-scale GMP production is thusfeasible.

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The references listed below, and all references cited in thespecification are hereby incorporated by reference in their entirety.

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What is claimed is:
 1. A composition comprising: an immunologicallyeffective amount of a recombinant modified vaccinia Ankara (rMVA) virus,wherein the rMVA virus comprises a fusion nucleotide sequence whichencodes an IEfusion CMV protein antigen, said fusion nucleotide sequencecomprising a nucleotide sequence encoding an Immediate-Early Gene-1(IE1) antigenic portion directly fused to a nucleotide sequence encodingan Immediate-Early Gene-2 (IE2) antigenic portion, wherein (i) thenucleotide sequence encoding the IE1 antigenic portion includes anucleotide sequence encoding IE1 exon 4 (IE1/e4); (ii) the nucleotidesequence encoding the IE2 antigenic portion is a nucleotide sequenceencoding IE2 exon 5 (IE2/e5); or (iii) both (i) and (ii).
 2. Thecomposition of claim 1, wherein the fusion nucleotide sequence comprisesSEQ ID NO:11.
 3. The composition of claim 1, further comprising anucleotide sequence which encodes at least one CMV antigen or acombination of antigens selected from the group consisting of HCMV-pp65,and glycoprotein B (gB).
 4. The composition of claim 1, wherein the rMVAvirus is genetically stable and maintains immunogenicity after serialpassage, and wherein the DNA sequence of the IE1 or IE2 gene and theexpression of the IE1 or IE2 gene is substantially unchanged over thetime of serial passage.
 5. The composition of claim 3, wherein thecomposition is produced by: a) constructing a transfer plasmid vectorcomprising a modified H5 (mH5) promoter operably linked to a DNAsequence encoding the IEfusion CMV protein antigen, wherein theexpression of said DNA sequence is under the control of the mH5promoter; b) generating the rMVA virus by transfecting the plasmidvector obtained from step a) into cells infected with wild type MVA; andc) identifying rMVA virus expressing the IEfusion CMV protein antigenusing one or more selection methods for serial passage; d) conductingserial passage; e) expanding an rMVA virus strain identified by step d);and f) purifying the rMVA virus strain from step e) to form thecomposition.
 6. The composition of claim 5, wherein the identificationof rMVA virus carrying the transfer plasmid vector is accomplished byone or more gene-in selection methods, one or more gene-out selectionmethods, or a combination of gene-in and gene-out selection methods. 7.The composition of claim 5, wherein the serial passage is at least 10passages.
 8. The composition of claim 5, wherein the transfer plasmidvector comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO:9 and SEQ ID NO:10.
 9. The composition of claim5 wherein the transfer plasmid vector comprises nucleotide sequences SEQID NO:9 and SEQ ID NO:10.
 10. A method of modifying an immune responsein a mammalian subject by administering the composition of claim 3 tothe subject.
 11. The method of claim 10, wherein the subject is a human.12. The method of claim 10, wherein the subject is a human stem celldonor or a human solid organ transplant donor.
 13. The method of claim10, wherein the subject is a human with an immunodeficiency disease or aheritable immunodeficiency and the subject is susceptible to infectionby human cytomegalovirus.
 14. The method of claim 10, wherein thesubject is a human subject who has received a stem cell transplant (HCT)or a solid organ transplant from a healthy donor.
 15. A method forproducing a genetically stable rMVA composition, comprising: a)constructing a transfer plasmid vector comprising a modified H5 (mH5)promoter operably linked to a DNA sequence encoding a heterologousforeign protein antigen, wherein the expression of said DNA sequence isunder the control of the mH5 promoter; b) generating rMVA virus bytransfecting the plasmid vector obtained from step a) into cellsinfected with wild type MVA; and c) identifying rMVA virus expressingthe heterologous foreign protein antigen using one or more selectionmethods for serial passage; d) conducting serial passage; e) expandingan rMVA virus strain identified by step d); and f) purifying the rMVAvirus strain from step e) to form the composition; wherein theexpression and immunogenicity of said foreign protein antigen are stableafter serial passage in the rMVA composition obtained from step f); andwherein the foreign protein antigen is an IEfusion CMV protein antigencomprising a nucleotide sequence encoding an Immediate-Early Gene-1(IE1) antigenic portion directly fused to a nucleotide sequence encodingan Immediate-Early Gene-2 (IE2) antigenic portion, wherein (i) thenucleotide sequence encoding the IE1 antigenic portion includes anucleotide sequence encoding IE1 exon 4 (IE1/e4); (ii) the nucleotidesequence encoding the IE2 antigenic portion is a nucleotide sequenceencoding IE2 exon 5 (IE2/e5); or (iii) both (i) and (ii).
 16. The methodof claim 15, wherein the IEfusion CMV protein antigen comprises anucleotide sequence of SEQ ID NO:11.
 17. The method of claim 16, whereinthe composition further comprises at least one CMV antigen selected fromthe group consisting of pp65, CMV pp150, glycoprotein B (gB) andantigenic fragments thereof, the UL128 complex or one or more membersthereof selected from the group consisting of UL128, UL130, UL131a, gH,and gL.
 18. The method of claim 15, wherein the identification of rMVAvirus carrying the transfer plasmid vector is accomplished by one ormore gene-in selection methods, one or more gene-out selection methods,or a combination of gene-in and gene-out selection methods.
 19. Themethod of claim 15, wherein the serial passage is at least 10 passages.20. A composition comprising an immunologically effective amount of anrMVA virus which is genetically stable after serial passage and producedby: a) constructing a transfer plasmid vector comprising a modified H5(mH5) promoter operably linked to a DNA sequence encoding an IEfusionCMV protein antigen comprising two or more antigenic portions ofImmediate-Early Gene-1 or Immediate-Early Gene-2, wherein the expressionof said DNA sequence is under the control of the mH5 promoter; b)generating rMVA virus by transfecting the plasmid vector obtained fromstep a) into cells infected with wild type MVA; and c) identifying rMVAvirus expressing the IEfusion CMV protein antigen using one or moreselection methods for serial passage; d) conducting serial passage; e)expanding an rMVA virus strain identified by step d); and f) purifyingthe rMVA virus strain from step e) to form the composition; wherein theexpression and immunogenicity of said foreign protein antigen are stableafter serial passage in the rMVA composition obtained from step f); andwherein the IEfusion CMV protein antigen comprises a nucleotide sequenceencoding an Immediate-Early Gene-1 (IE1) antigenic portion directlyfused to a nucleotide sequence encoding an Immediate-Early Gene-1 (IE2)antigenic portion, wherein (i) the nucleotide sequence encoding the IE1antigenic portion includes a nucleotide sequence encoding IE1 exon 4(IE1/e4); (ii) the nucleotide sequence encoding the IE2 antigenicportion is a nucleotide sequence encoding IE2 exon 5 (IE2/e5); or (iii)both (i) and (ii).
 21. A cytomegalovirus (CMV) composition comprising:an immunologically effective amount of a recombinant modified vacciniaAnkara (rMVA) virus, wherein the rMVA virus comprises a fusionnucleotide sequence which encodes an IEfusion CMV protein antigen, saidfusion nucleotide sequence comprising a nucleotide sequence encoding anImmediate-Early Gene-1 (IE1) antigenic portion directly fused to anucleotide sequence encoding an Immediate-Early Gene-2 (IE2) antigenicportion, wherein (i) the nucleotide sequence encoding the IE1 antigenicportion includes a nucleotide sequence encoding IE1 exon 4 (IE1/e4);(ii) the nucleotide sequence encoding the IE2 antigenic portion is anucleotide sequence encoding IE2 exon 5 (IE2/e5); or (iii) both (i) and(ii).
 22. A CMV composition comprising an immunologically effectiveamount of an rMVA virus produced by: a) constructing a transfer plasmidvector comprising a modified H5 (mH5) promoter operably linked to a DNAsequence encoding an IEfusion CMV protein antigen comprising two or moreantigenic portions of Immediate-Early Gene-1 or Immediate-Early Gene-2,wherein the expression of said DNA sequence is under the control of themH5 promoter; b) generating rMVA virus by transfecting the plasmidvector obtained from step a) into cells infected with wild type MVA; andc) identifying rMVA virus expressing the IEfusion CMV protein antigenusing one or more selection methods for serial passage; d) conductingserial passage; e) expanding an rMVA virus strain identified by step d);and f) purifying the rMVA virus strain from step e) to form thecomposition; and wherein the IEfusion CMV protein antigen comprises anucleotide sequence encoding an Immediate-Early Gene-1 (IE1) antigenicportion directly fused to a nucleotide sequence encoding anImmediate-Early Gene-1 (IE2) antigenic portion, wherein (i) thenucleotide sequence encoding the IE1 antigenic portion includes anucleotide sequence encoding IE1 exon 4 (IE1/e4); (ii) the nucleotidesequence encoding the IE2 antigenic portion is a nucleotide sequenceencoding IE2 exon 5 (IE2/e5); or (iii) both (i) and (ii).
 23. The methodof claim 13, wherein the immunodeficiency disease is HIV.